Altered OspA of Borrelia burgdorferi

ABSTRACT

Provided herein are OspA polypeptides from Lyme Disease-causing Borrelia having certain alteration(s). In one embodiment, the alteration(s) increase the conformational stability of the OspA polypeptide containing the alteration(s) while maintaining at least some of the antigenicity of the corresponding unaltered OspA polypeptide. In another embodiment, the altered OspA polypeptide has reduced cross-reactivity to hLFA-1, as compared to the corresponding unaltered OspA polypeptide.

RELATED APPLICATIONS

[0001] This application is a continuation of International ApplicationNo. PCT/US01/25852, which designated the United States, was published inEnglish and was filed on Aug. 17, 2001. This application also claims thebenefit of U.S. Provisional Application No. 60/226,484, filed on Aug.18, 2000. The entire teachings of both of these applications areincorporated herein by reference.

GOVERNMENT SUPPORT

[0002] The invention was supported, in whole or in part, by Grant2R01A137256-05A1 from the National Institute of Allergy and InfectiousDiseases. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Lyme disease (Lyme borreliosis) is the most common tick-borneinfectious disease in North America and Europe, and has been found inRussia, Japan, China and Australia. Lyme disease begins at the site of atick bite, producing a primary infection with spread of the organism tosecondary sites occurring during the course of infection. The causativebacterial agent of this disease is the spirochete Borrelia burgdorferi,which was first isolated and cultivated in 1982 (Burgdorferi, W. A. etal., Science 216: 1317-1319 (1982); Steere, A. R. et al., N. Engl. J.Med., 308:733-740 (1983)).

[0004] Three pathogenic genospecies of Borrelia, B. burgdorferi sensustricto (B. burgdorferi or B.b.s.s.), B. afzelii and B. garinii havebeen described (Baranton, G., et al., Int. J. Syst. Bacteriol.,42:378-383 (1992)). These are members of a species complex, B.burgdorferi sensu lato, which consists of at least 10 differentgenospecies (Piken, R. N. et al., J. Invest. Dermatol., 110:211-214(1998); Postic, D. et al., Int. J. Syst. Bacteriol., 44:743-752 (1994);Valsangiacomo, C. T. et al., Int. J. Syst. Bacteriol., 47:1-10 (1997)).The three genospecies, B. burgdorferi sensu stricto, B. afzelii and B.garinii, are all thought to be pathogenic and all are found in Europe.

[0005]B. burgdorferi has an outer membrane whose major proteinconstituents are the outer surface proteins A and B (OspA and OspB).OspA is a basic lipoprotein of approximately 31 kd, which is encoded ona large linear plasmid along with OspB, a basic lipoprotein ofapproximately 34 kd (Szczepanski, A., and J. L. Benach, Microbiol. Rev.,55:21 (1991)). The immune response to these outer surface proteins tendsto occur late in the disease, if at all (Craft, J. E. et al., J. ClinInvest. 78:934-939 (1986); Dattwyler, R. J. and B. J. Luft, Rheum. Clin.North Am., 15:727-734 (1989)). Furthermore, patients acutely andchronically infected with B. burgdorferi respond variably to thedifferent antigens, including OspA, OspB, OspC, OspD, p39, p41 and p93.

[0006] Currently, Lyme Disease is treated with a range of antibiotics,e.g., tetracyclines, penicillin and cephalosporins. However, suchtreatment is not always successful in clearing the infection. Treatmentis often delayed due to improper diagnosis with the deleterious effectthat the infection proceeds to a chronic condition, where treatment withantibiotics is often not useful. One of the factors contributing todelayed treatment is the lack of effective diagnostic tools.

[0007] Vaccines against Lyme borreliosis have been attempted. However, avaccine that consists of recombinant OspA may require frequent boosterimmunizations. An additional concern of OspA-based vaccines is therecent identification of a putative autoreactive OspA domain with a highdegree of similarity to a region of human leukocyte function-associatedantigen-1 (hLFA-1) (Gross, D. M. et al., Science, 281: 703-706 (1998)).

[0008] Therefore, it should be advantageous to develop modified OspAproteins having decreased cross-reactivity to hLFA-1 in order to reducepotential side effects of an OspA vaccine. Development of OspA proteinswith decreased hLFA-1 cross-reactivity that maintain or have increasedimmunoreactivity to more than one member of the Borrelia complex wouldalso be desirable. To be useful as vaccines, the conformations of thesemodified proteins must be sufficiently stable to retain certain OspAstructural features that are required to elicit a protective immuneresponse. OspA proteins with these features would allow for improvementsin diagnosis and/or vaccination against all, or most, of the Borreliathat cause Lyme Disease.

[0009] Analysis of the immune status of OspA immunized individualsrevealed that the overall quantitative response is not predictive ofprotection, but rather the reactivity with a specific epitope of theOspA lipoprotein directly correlates to protective immunity. Theanti-OspA monoclonal antibody, LA-2 (Kramer et al., 1990) defines anepitope of the lipoprotein that is apparently necessary for protectiveimmunity after OspA vaccination. For instance, passive immunization ofmice with this antibody leads to protection against infection with thespirochete (Schaible et al., 1993). In addition, immunization of miceand canines with OspA resulting in significant titers of LA-2 equivalentserum antibody accurately predicts protection from tick transmission ofinfection (Golde, 1997). Insufficient levels of LA-2 equivalent antibodyresult in a lack of protection in the face of high serum antibody titersto OspA (Johnson et al., 1995).

SUMMARY OF THE INVENTION

[0010] The present invention is drawn to altered forms of OspA fromBorrelia burgdorferi that have increased conformational stability whilemaintaining at least some of the antigenicity of wild type OspA. In someembodiments, the altered OspA polypeptide has decreased cross-reactivitywith hLFA-1, as compared to the corresponding unaltered OspApolypeptide. The altered OspA polypeptides can comprise almost all oronly a portion of the native OspA polypeptide. In some embodiments, thealtered OspA polypeptide can be part of a cocktail which includes one ormore other proteins, such as, for example, other Borrelia burgdorferipolypeptides including OspA, OspB, OspC, OspD, p93 and p41. In otherembodiments, the altered OspA polypeptide can be part of a chimericprotein, such as those described in U.S. Pat. No. 6,248,562, the entireteachings of which are incorporated herein by reference.

[0011] In one embodiment, the altered OspA polypeptides of the presentinvention comprise an amino acid sequence of OspA protein from Borreliaburgdorferi from about residue 139 to about residue 273, wherein thesequence includes at least one alteration selected from the groupconsisting of: residue 139 changed to methionine, residue 160 changed totyrosine, residue 189 changed to methionine and combinations thereof. Inother embodiments, the altered OspA polypeptides of the presentinvention comprise an amino acid sequence of OspA protein from Borreliaburgdorferi from about residue 131 to about residue 273 or from aboutresidue 17 to about residue 273. The OspA polypeptides of the presentinvention can comprise longer or shorter fragments of OspA protein. Thenumbering of the residues corresponds to the numbering of SEQ ID NO: 7(OspA from B31).

[0012] In another embodiment, the OspA polypeptides of the presentinvention comprise an amino acid sequence of OspA protein from Borreliaburgdorferi from about residue 160 to about residue 170, wherein thesequence includes at least two alterations selected from the groupconsisting of: residue 165 changed to phenylalanine, residue 166 changedto threonine and residue 170 changed to lysine. In yet anotherembodiment, the OspA polypeptides of the present invention comprise anamino acid sequence of OspA protein from a sensu stricto strain ofBorrelia burgdorferi from about residue 160 to about residue 170,wherein the sequence includes at least one alteration selected from thegroup consisting of: residue 165 changed to phenylalanine, residue 166changed to threonine, residue 170 changed to lysine and combinationsthereof. In other embodiments, the altered OspA polypeptides of thepresent invention comprise an amino acid sequence of OspA protein fromBorrelia burgdorferi from about residue 150 to about residue 180 or fromabout residue 17 to about residue 273. The OspA polypeptides of thepresent invention can comprise longer or shorter fragments of OspAprotein. The numbering of the residues corresponds to the numbering ofSEQ ID NO: 7.

[0013] The polypeptides of the present invention include polypeptidesselected from the group consisting of: SEQ ID NO: 96, 98, 100, 102, 104,106, 108, 110, 112, 114 and 116.

[0014] The present invention is also drawn to polynucleotides encodingthe amino acid sequences described herein, such as polynucleotidesencoding OspA polypeptides from Borrelia burgdorferi from about residue131 to about residue 273, wherein the sequence encodes at least onealteration selected from the group consisting of: codon 139 encodingmethionine, codon 160 encoding tyrosine, codon 189 encoding methionineand combinations thereof. The polynucleotide encoding OspA polypeptidesof the present invention can encode longer or shorter fragments of OspAprotein. The numbering of the residues corresponds to the numbering ofSEQ ID NO: 7.

[0015] In another embodiment, the polynucleotide encodes an amino acidsequence of an OspA polypeptide from Borrelia burgdorferi from aboutresidue 160 to about residue 170, wherein the sequence encodes at leastone alteration selected from the group consisting of: codon 165 encodingphenylalanine, codon 166 encoding threonine, codon 170 encoding lysineand combinations thereof. The polynucleotides which encode OspApolypeptides of the present invention can encode longer or shorterfragments of OspA protein. The numbering of the residues corresponds tothe numbering of SEQ ID NO: 7.

[0016] The polynucleotides of the present invention include apolynucleotide selected from the group consisting of: SEQ ID NO: 95, 97,99, 101, 103, 105, 107, 109, 111, 113 and 115.

[0017] The present invention is also drawn to a method of generating analtered Borrelia burgdorferi OspA polypeptide with increasedconformational stability, as compared to the corresponding unalteredBorrelia burgdorferi OspA polypeptide. The method comprises selecting apolynucleotide encoding a Borrelia burgdorferi OspA polypeptide thatincludes at least one of residues 139, 160 and 189, wherein thenumbering corresponds to the numbering of SEQ ID NO: 7. Thepolynucleotide is altered such that at least one of the followingalterations is present: residue 139 is changed to methionine, residue160 is changed to tyrosine and residue 189 is changed to methionine or acombination thereof. In one embodiment, both the alteration at residue160 and the alteration at 189 is made. In another embodiment, thealterations at all three residues are made. The altered polynucleotideis expressed, thereby generating an altered Borrelia burgdorferi OspApolypeptide with increased conformational stability, as compared to thecorresponding unaltered Borrelia burgdorferi OspA polypeptide.

[0018] In another embodiment, the present invention is drawn to a methodof generating an altered Borrelia burgdorferi OspA polypeptide withreduced cross-reactivity with an hLFA-1 molecule, as compared to thecorresponding unaltered Borrelia burgdorferi OspA polypeptide. Themethod comprises selecting a polynucleotide encoding an OspA polypeptidefrom Borrelia burgdorferi that includes at least one of residues 165,166 and 170, wherein the numbering corresponds to the numbering of SEQID NO: 7. The polynucleotide is altered such that at least onealteration from the following list is present: residue 165 is changed tophenylalanine, residue 166 is changed to threonine and residue 170 ischanged to lysine or combination thereof. The altered polynucleotide isexpressed, thereby generating an altered Borrelia burgdorferi OspApolypeptide with reduced cross-reactivity with the hLFA-1 molecule, ascompared to the corresponding unaltered Borrelia burgdorferi OspApolypeptide.

[0019] The present invention is also drawn to an expression vector whichcomprises an isolated DNA encoding an altered Borrelia OspA protein. Thepresent invention also encompasses a host cell which comprises arecombinant nucleic acid that encodes an altered OspA protein asdescribed herein.

[0020] The present invention is also drawn to a method of delivering thealtered Borrelia OspA polypeptides described herein. In one embodiment,the method comprises administering the altered OspA polypeptide in aphysiologically-acceptable carrier to an individual. As a result of theadministration of the altered OspA protein, the individual develops atleast some immune response to the protein. As an example, the individualgenerates a humoral immune response, wherein antibodies that recognizeat least a portion of said polypeptide are produced by the individual.In a preferred embodiment, the individual generates an immunoprotectiveresponse, for example, by generating antibodies that recognize the LA-2epitope.

[0021] The present invention is also drawn to a method of delivering anucleic acid which encodes an altered OspA polypeptide described herein.In one embodiment, the method comprises administering the nucleic acidin a physiologically-acceptable carrier to an individual. As a result ofthe administration of the nucleic acid, the altered OspA polypeptide isat least transiently expressed and the individual develops at least someimmune response, preferably an immunoprotective response to the alteredOspA protein encoded by the nucleic acid. As an example, the individualgenerates a humoral immune response, wherein antibodies that recognizeat least a portion of the altered OspA polypeptide produced from thenucleic acid are produced by the individual. In a preferred embodiment,the individual generates an immunoprotective response, for example, bygenerating antibodies that recognize the LA-2 epitope.

[0022] The invention also encompasses methods of using the proteinsdescribed herein in diagnostic assays. In one embodiment, the method canbe used to detect the presence of OspA specific antibodies in a hostsample of interest. The method comprises contacting a host sample ofinterest with the altered protein, under conditions wherein antibodies,if present in the host sample, bind to the altered protein, formingantigen-antibody complexes. The antigen-antibody complexes are thendetected using standard methods known in the art.

[0023] The present invention is also drawn to a diagnostic kitcomprising the altered polypeptides described herein. The kit comprisesan altered Borrelia burgdorferi OspA protein as described herein. Thekit also includes reagents for detecting antibody-antigen complexes thatare formed between the OspA altered protein and antibodies that arepresent in the user-supplied host sample.

[0024] As a result of the present invention, OspA proteins, or fragmentsthereof, having either increased conformational stability whilemaintaining at least some antigenicity, or having reducedcross-reactivity to hLFA-1, are available for use in research, vaccinesand/or diagnostic assays. Furthermore, as a result of the presentinvention, nucleic acids encoding OspA polypeptides having reducedcross-reactivity with hLFA-1 are available for research and vaccines.The altered OspA polypeptides of the present invention are expected toallow for improved vaccines having fewer side effects.

[0025] For a better understanding of the present invention together withother and further objects, reference is made to the followingdescription, taken together with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

[0027]FIG. 1 summarizes peptides and antigenic domains localized byproteolytic and chemical fragmentation of OspA.

[0028]FIG. 2 is a comparison of the antigenic domains depicted in FIG.1, for OspA in nine strains of B. burgdorferi.

[0029]FIG. 3 is a graph depicting a plot of weighted polymorphism versusamino acid position among 14 OspA variants. The marked peaks are: a)amino acids 132-145; b) amino acids 163-177; c) amino acids 208-221. Thelower dotted line at polymorphism value 1.395 demarcates statisticallysignificant excesses of polymorphism at p=0.05. The upper dotted line at1.520 is the same, except that the first 29 amino acids at themonomorphic-terminus have been removed from the original analysis.

[0030]FIG. 4 depicts the amino acid alignment of residues 200 through220 for OspAs from strains B31 and K48 as well as for the site-directedmutants 613, 625, 640, 613/625, and 613/640. Arrow indicates Trp216.Amino acid changes are underlined.

[0031]FIG. 5 depicts a phylogenetic tree for strains of Borreliadescribed in Table I. The strains are as follows: 1=B31; 2=Pka1; 3=ZS7;4=N40; 5=25015; 6=K48; 7=DK29; 8=PHei; 9=Ip90; 10=PTrob; 11=ACAI;12=PGau; 13=Ip3; 14=PBo; 15=Pko.

[0032]FIGS. 6A and 6B depict the nucleic acid sequence of OspA-B31 (SEQID NO: 6), and the encoded protein sequence (SEQ ID NO: 7).

[0033]FIGS. 7A, 7B and 7C depict the nucleic acid sequence of OspA-K48(SEQ ID NO: 8), and the encoded protein sequence (SEQ ID NO: 9).

[0034]FIGS. 8A, 8B and 8C depict the nucleic acid sequence of OspA-PGau(SEQ ID NO: 10), and the encoded protein sequence (SEQ ID NO: 11).

[0035]FIGS. 9A and 9B depict the nucleic acid sequence of an OspA gene(SEQ ID NO: 127 and its encoded protein sequence (SEQ ID NO: 128).

[0036]FIGS. 10A, 10B and 10C depict the nucleic acid sequence of theOspA-K48/OspA-PGau chimer (SEQ ID NO: 28) and the encoded chimericprotein sequence (SEQ ID NO: 29).

[0037]FIGS. 11A, 11B and 11C depict the nucleic acid sequence of theOspA-B31/OspA-PGau chimer (SEQ ID NO: 30) and the encoded chimericprotein sequence (SEQ ID NO: 31).

[0038]FIGS. 12A and 12B depict the nucleic acid sequence of theOspA-B31/OspA-K48 chimer (SEQ ID NO: 32) and the encoded chimericprotein sequence (SEQ ID NO: 33).

[0039]FIGS. 13A, 13B and 13C depict the nucleic acid sequence of theOspA-B31/OspA-25015 chimer (SEQ ID NO: 34) and the encoded chimericprotein sequence (SEQ ID NO: 35).

[0040]FIGS. 14A, 14B and 14C depict the nucleic acid sequence of theOspA-K48/OspA-B31/OspA-K48 chimer (SEQ ID NO: 36) and the encodedchimeric protein sequence (SEQ ID NO: 37).

[0041]FIGS. 15A, 15B and 15C depict the nucleic acid sequence of theOspA-B31/OspA-K48/OspA-B31/OspA-K48 chimer (SEQ ID NO: 38) and theencoded chimeric protein sequence (SEQ ID NO: 39).

[0042]FIGS. 16A, 16B and 16C depict the nucleic acid sequence of theOspA-B31/OspB-B31 chimer (SEQ ID NO: 40) and the encoded chimericprotein sequence (SEQ ID NO: 41).

[0043]FIGS. 17A, 17B, 17C, 17D, 17E, 17F, 17G, 17H, 17I, 17J, 17K, 17L,17M, 17N, 17O, and 17P, depict an alignment of the nucleic acidsequences for OspA-B31 (SEQ ID NO: 6), OspA-pKa1 (SEQ ID NO: 42),OspA-N40 (SEQ ID NO: 43), OspA-ZS7 (SEQ ID NO: 44), OspA-25015 (SEQ IDNO: 12), OspA-pTrob (SEQ ID NO: 45), OspA-K48 (SEQ ID NO: 8), OspA-Hei(SEQ ID NO: 46), OspA-DK29 (SEQ ID NO: 21), OspA-Ip90 (SEQ ID NO: 22),OspA-pBo (SEQ ID NO: 23), OspA-Ip3 (SEQ ID NO: 24), OspA-Pko (SEQ ID NO:25), OspA-ACAI (SEQ ID NO: 26), and OspA-PGau (SEQ ID NO: 10). Nucleicacids which are identical to those in the lead nucleic acid sequence(here, OspA-B31) are represented by a period (.); differing nucleicacids are shown in lower case letters.

[0044]FIGS. 18A and 18B depict the nucleic acid sequence of theOspA-Tro/OspA-Bo chimer (SEQ ID NO: 47) and the encoded chimeric proteinsequence (SEQ ID NO: 48).

[0045]FIGS. 19A and 19B depict the nucleic acid sequence of theOspA-PGau/OspA-Bo chimer (SEQ ID NO: 49) and the encoded chimericprotein sequence (SEQ ID NO: 50).

[0046]FIGS. 20A and 20B depict the nucleic acid sequence of theOspA-B31/OspA-PGau/OspA-B31/OspA-K48 chimer (SEQ ID NO: 53) and theencoded chimeric protein sequence (SEQ ID NO: 54).

[0047]FIG. 21A and 21B depict the nucleic acid sequence of theOspA-PGau/OspA-B31/OspA-K48 chimer (SEQ ID NO: 51) and the encodedchimeric protein sequence (SEQ ID NO: 52).

[0048]FIG. 22 is a bar graph showing the reactivity (as measured byELISA) of sera from mice immunized with the indicated Borrelia protein(OspA or OspC) or recombinant chimeric protein (OspC2-OspA)(X-axis)against the indicated OspA or OspC antigens (legend) from the strain B31(Borrelia burgdorferi sensu stricto).

[0049]FIG. 23 is a bar graph showing the reactivity (as measured byELISA) of sera from mice immunized with the indicated Borrelia protein(OspA or OspC) or recombinant chimeric protein (OspC2-OspA)(X-axis)against the indicated OspA or OspC antigens (legend) from the strain B31(Borrelia burgdorferi sensu stricto). For the ELISA results to the B31OspA antigen, a purified fragment of B31 OspA (amino acids 18-139) wasadded in excess to the sera so that the detected immune response wasspecific for the C-terminal region of OspA.

[0050]FIG. 24 is a bar graph showing the reactivity of sera from miceimmunized with the indicated Borrelia chimeric protein (lipOspA/Bo,lipOspAB/P or OspC-OspAB/P)(X-axis) against the indicated OspA antigens(legend) from strains B31 (Borrelia burgdorferi sensu stricto), K48(Borrelia garinii) and Pgau (Borrelia afzelii).

[0051]FIG. 25 is a bar graph showing the reactivity of sera from miceimmunized with the indicated Borrelia chimeric protein (lipOspAP/Bo,lipOspAB/P or OspC-OspAB/P)(X-axis) against the indicated OspA (legend)from strains B31 (Borrelia burgdorferi sensu stricto), K48 (Borreliagarinii) and Pgau (Borrelia afzelii). In all cases, a purified fragmentof B31 OspA (amino acids 18-139) was added in excess to the sera so thatthe detected immune response is specific for the C-terminal region ofOspA.

[0052]FIG. 26 is a bar graph showing the reactivity of sera from miceimmunized with the indicated Borrelia chimeric protein (OspCB31-OspAB31,OspC2-OspAB31 or lip OspC-B31)(X-axis) against the indicated OspCantigen (legend) from the strain B31 (Borrelia burgdorferi sensustricto).

[0053]FIG. 27 is a bar graph showing the reactivity of sera from miceimmunized with the indicated Borrelia chimeric protein (OspCB31-OspAB31,OspC2-OspAB31 or Lip OspA K/T)(X-axis) against the indicated OspAantigens (legend) from strains B31 (Borrelia burgdorferi sensu stricto),K48 (Borrelia garinii) and Pgau (Borrelia afzelii).

[0054]FIG. 28 is a bar graph showing the reactivity of sera from miceimmunized with the indicated Borrelia chimeric protein (OspCB31-OspAB/P,OspCB31-OspABPBP or OspCB31-OspAB31)(X-axis) against the indicated OspAantigens (legend) from strains B31 (Borrelia burgdorferi sensu stricto),K48 (Borrelia garinii) and Pgau (Borrelia afzelii).

[0055]FIG. 29 is a bar graph showing the reactivity of sera from miceimmunized with the indicated Borrelia chimeric protein (OspCB31-OspAB/P,OspCB31-OspABPBP or OspCB31-OspAB31)(X-axis) against the indicated OspA(legend) from strains B31 (Borrelia burgdorferi sensu stricto), K48(Borrelia garinii) and Pgau (Borrelia afzelii). In all cases, a purifiedfragment of B31 OspA (amino acids 18-139) was added in excess to thesera so that the detected immune response is specific for the C-terminalregion of OspA.

[0056]FIGS. 30A, 30B and 30C depict the nucleic acid sequence of theOspC-B31 (bp 55-633)/OspA-B31 (bp 52-822) chimer (SEQ ID NO: 55) and theencoded chimeric protein sequence (SEQ ID NO: 56).

[0057]FIGS. 31A, 31B and 31C depict the nucleic acid sequence of theOspC-B31 (bp 55-624)/OspA-B31 (bp 52-822) chimer (SEQ ID NO: 57) and theencoded chimeric protein sequence (SEQ ID NO: 58).

[0058]FIGS. 32A, 32B and 32C depict the nucleic acid sequence of theOspC-C2 (bp 55-612)/OspA-B31 (bp 52-822) chimer (SEQ ID NO: 59) and theencoded chimeric protein sequence (SEQ ID NO: 60).

[0059]FIGS. 33A, 33B, and 33C depict the nucleic acid sequence of theOspC-B31 (bp 55-633)/OspA-B31 (bp 52-651)/OspA-K48 (bp 652-820) chimer(SEQ ID NO: 61) and the encoded chimeric protein sequence (SEQ ID NO:62).

[0060]FIGS. 34A, 34B and 34C depict the nucleic acid sequence of theOspC-C2 (bp 55-612)/OspA-B31 (bp 52-651)/OspA-K48 (bp 652-820) chimer(SEQ ID NO: 63) and the encoded chimeric protein sequence (SEQ ID NO:64).

[0061]FIGS. 35A, 35B and 35C depict the nucleic acid sequence of theOspC-B31 (bp 55-633)/OspA-B31 (bp 52-651)/OspA-Pko (bp 652-820) chimer(SEQ ID NO: 65) and the encoded chimeric protein sequence (SEQ ID NO:66).

[0062]FIGS. 36A, 36B and 36C depict the nucleic acid sequence of theOspC-C2 (bp 55-612)/OspA-B31 (bp 52-651)/OspA-Pko (bp 652-820) chimer(SEQ ID NO: 67) and the encoded chimeric protein sequence (SEQ ID NO:68).

[0063]FIGS. 37A, 37B and 37C depict the nucleic acid sequence of theOspC-B31 (bp 55-633)/OspA-K48 (bp 52-654)/OspA-Tro (bp 655-819) chimer(SEQ ID NO: 69) and the encoded chimeric protein sequence (SEQ ID NO:70).

[0064]FIGS. 38A, 38B and 38C depict the nucleic acid sequence of theOspC-C2 (bp 55-612)/OspA-K48 (bp 52-654)/OspA-Tro (bp 655-819) chimer(SEQ ID NO: 71) and the encoded chimeric protein sequence (SEQ ID NO:72).

[0065]FIGS. 39A, 39B, 39C depict the nucleic acid sequence of theOspC-C12 (bp 55-612)/OspA-B31 (bp 88-450)/OspA-Pko (bp 451-537)/OspA-B31(bp 538-822) chimer (SEQ ID NO: 73) and the encoded chimeric proteinsequence (SEQ ID NO: 74).

[0066]FIGS. 40A, 40B and 40C depict the nucleic acid sequence of theOspC-Pko (bp 55-639)/OspA-B31 (bp 88-450)/OspA-Pko (bp 451-537)/OspA-B31(bp 538-651)/OspA-K48 (bp 652-825) chimer (SEQ ID NO: 75) and theencoded chimeric protein sequence (SEQ ID NO: 76).

[0067]FIGS. 41A, 41B and 41C depict the nucleic acid sequence of theOspC-Tro (bp 55-624)/OspA-B31 (bp 88-450)/OspA-Pko (bp 451-537)/OspA-B31(bp 538-651)/OspA-Pko (bp 652-822) chimer (SEQ ID NO: 77) and theencoded chimeric protein sequence (SEQ ID NO: 78).

[0068]FIGS. 42A and 42B depict the nucleic acid sequence of the OspC-B31(bp 55-633)/OspA-B31 (bp 394-820) chimer (SEQ ID NO: 79) and the encodedchimeric protein sequence (SEQ ID NO: 80).

[0069]FIGS. 43A and 43B depict the nucleic acid sequence of the OspC-B31(bp 55-631)/OspA-B31 (bp 394-651)/OspA-K48 (bp 652-820) chimer (SEQ IDNO: 81) and the encoded chimeric protein sequence (SEQ ID NO: 82).

[0070]FIGS. 44A and 44B depict the nucleic acid sequence of the OspC-B31(bp 55-633)/OspA-B31 (bp 394-651)/OspA-Pko (bp 652-820) chimer (SEQ IDNO: 83) and the encoded chimeric protein sequence (SEQ ID NO: 84).

[0071]FIGS. 45A and 45B depict the nucleic acid sequence of the OspC-B31(bp 55-633)/OspA-K48 (bp 394-654)/OspA-Tro (bp 655-819) chimer (SEQ IDNO: 85) and the encoded chimeric protein sequence (SEQ ID NO: 86).

[0072]FIGS. 46A, 46B and 46C depict the nucleic acid sequence of theOspC-B31 (bp 55-633)/OspA-B31 (bp 88-450)/OspA-Pko (bp 451-537)/OspA-B31(bp 541-651)/OspA-Pko (bp 652-822) chimer (SEQ ID NO: 87) and theencoded chimeric protein sequence (SEQ ID NO: 88).

[0073]FIGS. 47A, 47B, and 47C depict the nucleic acid sequence of theOspC-C2 (bp 55-612)/OspA-B31 (bp 88-450)/OspA-Pko (bp 451-537)/OspA-B31(bp 541-651)/OspA-Pko (bp 652-822) chimer (SEQ ID NO: 89) and theencoded chimeric protein sequence (SEQ ID NO: 90).

[0074]FIGS. 48A and 48B depict the nucleic acid and encoded proteinsequence of an R139M altered OspA (SEQ ID NO: 95 and 96).

[0075]FIGS. 49A and 49B depict the nucleic acid and encoded proteinsequence of an E160Y altered OspA (SEQ ID NO: 97 and 98).

[0076]FIGS. 50A and 50B depict the nucleic acid and encoded proteinsequence of an R139M, E160Y altered OspA (SEQ ID NO: 99 and 100).

[0077]FIGS. 51A and 51B depict the nucleic acid and encoded proteinsequence of an E160Y altered OspA (SEQ ID NO: 101 and 102).

[0078]FIGS. 52A and 52B depict the nucleic acid and encoded proteinsequence of an R139M, E160Y, K189M altered OspA (SEQ ID NO: 103 and104).

[0079]FIGS. 53A and 53B depict the nucleic acid and encoded proteinsequence of an Y165F altered OspA (SEQ ID NO: 105 and 106).

[0080]FIGS. 54A and 54B depict the nucleic acid and encoded proteinsequence of an Y165F, V166T altered OspA (SEQ ID NO: 107 and 108).

[0081]FIGS. 55A and 55B depict the nucleic acid and encoded proteinsequence of a V166T altered OspA (SEQ ID NO: 109 and 110).

[0082]FIGS. 56A and 56B depict the nucleic acid and encoded proteinsequence of a V166T, T170K altered OspA (SEQ ID NO: 111 and 112).

[0083]FIGS. 57A and 57B depict the nucleic acid and encoded proteinsequence of an Y165F, V166T, T170K altered OspA (SEQ ID NO: 113 and114).

[0084]FIGS. 58A and 58B depict the nucleic acid and encoded proteinsequence of an R139M, E160Y, K189M, Y165F, V166T, T170K altered OspA(SEQ ID NO: 115 and 116).

DETAILED DESCRIPTION OF THE INVENTION

[0085] A description of preferred embodiments of the invention follows.

[0086] The present invention is drawn to altered forms of OspA fromBorrelia burgdorferi that have increased conformational stability whilemaintaining antigenicity, as indicated, for example, by the ability tobe bound by the LA-2 monoclonal antibody. In some embodiments, thealtered OspA polypeptides also have decreased cross-reactivity withhLFA-1. The altered OspA polypeptides can comprise all (with theexception of the alterations described herein) or a portion, such as theC-terminal portion, of a wild type OspA polypeptide. Applicants havefound that some forms of the OspA protein, such as truncated versions ofOspA, do not elicit a strong immunoprotective response when administeredto an animal even when the OspA polypeptide has the immunoprotectiveLA-2 epitope sequence.

[0087] The structure for recombinant OspA has been determined to 1.95 Åresolution in a binary complex with the Fab fragment of thenonprotective mouse mAb184.1, which is reactive with the OspA-terminus(Li et al., Proc. Natl. Acad. Sci. U.S.A. 94:3584-3589 (1997)). The OspApolypeptide folds into 21 consecutive antiparallel β-strands followed bya C-terminal α-helix. The structure is conveniently described as twodiscrete folded domains, an N-terminal sandwich domain and a C-terminalbarrel domain, connected by a long central β-sheet. One set of alteredpolypeptides described herein is designed to remove buried chargesand/or salt bridges in the OspA C-terminal portion and replace them withresidues that promote hydrophobic interactions.

[0088] Accordingly, in one embodiment, the altered OspA polypeptides ofthe present invention comprise altered OspA protein or polypeptides fromBorrelia burgdorferi from about residue 139 to about residue 273,wherein the sequence includes at least one alteration selected from thegroup consisting of: residue 139 changed to methionine, residue 160changed to tyrosine, residue 189 changed to methionine and combinationsthereof. The numbering of the residues corresponds to the numbering ofSEQ ID NO: 7. In one embodiment, the altered OspA polypeptide has atyrosine at residue position 160 and a methionine at residue position189. These alterations have been found to stabilize the conformation ofthe immunoprotective LA-2 epitope in LA-2-containing OspA polypeptides.In another embodiment, the altered OspA polypeptide has a methionine atresidue position 139, a tyrosine at residue position 160 and amethionine at residue position 189. In other embodiments, the alteredOspA polypeptides have both increased conformational stability andreduced cross-reactivity to the hLFA-1 protein.

[0089] For the alterations at positions 139, 160 and 189, the alteredOspA sequence can be from any Lyme borreliosis strain of Borreliaburgdorferi, such as strains from Borrelia burgdorferi sensu stricto,Borrelia afzelii and Borrelia garinii. Strains of Borrelia burgdorferiare well known to those of skill in the art. For example, strains ofBorrelia burgdorferi sensu stricto include B31, strains of Borreliaafzelii include Pgau and Pko and strains of Borrelia garinii includeK48.

[0090] In one embodiment, the OspA polypeptides of the present inventioncomprise an amino acid sequence of OspA protein from Borreliaburgdorferi from about residue 160 to about residue 170, wherein thesequence includes at least two alterations selected from the groupconsisting of: residue 165 changed to phenylalanine, residue 166 changedto threonine and residue 170 changed to lysine, wherein the numbering ofthe residues corresponds to the numbering of SEQ ID NO: 7. In anotherembodiment, the OspA polypeptides of the present invention comprise anamino acid sequence of OspA protein from a sensu stricto strain ofBorrelia burgdorferi from about residue 160 to about residue 170,wherein the sequence includes at least one alteration selected from thegroup consisting of: residue 165 changed to phenylalanine, residue 166changed to threonine, residue 170 changed to lysine and combinationsthereof. The numbering of the residues corresponds to the numbering ofSEQ ID NO: 7. In another embodiment, the altered OspA polypeptide has aphenylalanine at residue position 165 and a threonine at residueposition 166. In still another embodiment, the altered OspA polypeptidehas a phenylalanine at residue position 165, a threonine at residueposition 166 and a lysine at residue position 170. In yet anotherembodiment, the altered OspA polypeptide includes all of the alterationsdescribed herein. In this embodiment, the altered OspA polypeptide has amethionine at residue position 139, a tyrosine at residue position 160,a methionine at residue position 189, a phenylalanine at residueposition 165, a threonine at residue position 166 and a lysine atresidue position 170.

[0091] This invention also pertains to polypeptides comprising SEQ IDNO: 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, or 116. The alteredOspA polypeptides of the invention can be partially or substantiallypurified (e.g., purified to homogeneity), and/or substantially free ofother proteins.

[0092] The present invention is also drawn to polynucleotides encodingthe amino acid sequences described herein. As defined herein, the term“polynucleotide” refers to a nucleotide multimer or oligomer which iscomposed of deoxyribonucleotides or ribonucleotides, or a combinationthereof, having from a few, e.g., 2-20, to many, e.g., 20 to severalthousand or more, nucleotides. As such, polynucleotides include nucleicacids of any length and further encompass both naturally-occurring andsynthetic oligonucleotides and polynucleotides.

[0093] The polynucleotides of the present invention includepolynucleotides encoding OspA polypeptides from Borrelia burgdorferifrom about residue 139 to about residue 189, wherein the sequenceencodes at least one alteration selected from the group consisting of:codon 139 encoding methionine, codon 160 encoding tyrosine, codon 189encoding methionine and combinations thereof. The numbering of theresidues corresponds to the numbering of SEQ ID NO: 7. As describedabove for the polypeptides, in the case of alterations at positions 139,160 and 189, the polynucleotide encoding the altered OspA sequence canbe from any Lyme borreliosis strain of Borrelia burgdorferi.

[0094] In another embodiment, the polynucleotide encodes an amino acidsequence of an OspA polypeptide from Borrelia burgdorferi from aboutresidue 160 to about residue 170, wherein the sequence encodes at leastone alteration selected from the group consisting of: codon 165 encodingphenylalanine, codon 166 encoding threonine, codon 170 encoding lysineand combinations thereof. The numbering of the residues corresponds tothe numbering of SEQ ID NO: 7.

[0095] The polynucleotides of the present invention includepolynucleotides selected from the group consisting of: SEQ ID NO: 95,97, 99, 101, 103, 105, 107, 109, 111, 113 and 115.

[0096] The altered OspA polypeptides of the present invention can bederived from OspA molecules comprising fragments, derivatives, analogs,variants and mutants of the OspA protein (modified OspA) or can befragmented, derivatized, or otherwise altered after having thealterations described herein inserted. These modified OspA moleculespossess OspA antigenic activity.

[0097] The present invention is also drawn to a method of generating analtered Borrelia burgdorferi OspA polypeptide with increasedconformational stability, as compared to the corresponding unalteredBorrelia burgdorferi OspA polypeptide. The method comprises selecting apolynucleotide encoding a Borrelia burgdorferi OspA polypeptide thatincludes residues 139, 160 and 189, wherein the numbering corresponds tothe numbering of SEQ ID NO: 7. The polynucleotide is altered such thatresidue 139 is methionine, residue 160 is tyrosine or residue 189 ismethionine or combination thereof. The altered polynucleotide isexpressed, thereby generating an altered Borrelia burgdorferi OspApolypeptide with increased conformational stability compared to thecorresponding unaltered Borrelia burgdorferi OspA polypeptide. Methodsof altering a polynucleotide are described below and in theExemplification and are well known to those of skill in the art. Methodsof expressing the altered polypeptides of the invention are alsodescribed below and in the Exemplification and are well known to thoseof skill in the art.

[0098] Residues 165-173 on β-strand 13 of OspA have been implicated ininduction of Lyme-related arthritis (Gross D. M. et al., Science281:703-706 (1998)). This region has homology to residues 332-340 ofhLFA-1, suggesting that this protein has a cross-reactive T cell epitope(YVLEGTLTA-B31 (SEQ ID NO: 129 and YVIEGTSKQ-hLFA-1 (SEQ ID NO: 130),respectively). Although B. burgdorferi sensu stricto is generallybelieved to be more arthritogenic that other Borrelia strains, a recentstudy of ospA alleles in synovial fluid of patients with Lyme arthritisindicates that B. garinii and B. afzelii may also cause arthritis(Eiffert, L. F. et al., Scand. J. Infect. Dis. 30:265-268 (1998)).

[0099] One way to eliminate the cross-reactive sequence is to replacethe β-13 region of OspA-B31 (YVLEGTLTA (SEQ ID NO: 129)) with ananalogous region from a strain that does not possess the same sequence,such as that from a B. Afzelii strain, e.g., Pgau or Pko (U.S. PatentApplication entitled “Recombinant Constructs of Borrelia burgdorferi” byLuft et al., filed on Aug. 7, 2001, the entire teachings of which areincorporated herein by reference).

[0100] Accordingly, in another embodiment, the present invention isdrawn to a method of generating an altered Borrelia burgdorferi OspApolypeptide with reduced cross-reactivity with the hLFA-1 molecule, ascompared to the corresponding unaltered Borrelia burgdorferi OspApolypeptide. The method comprises selecting a polynucleotide encoding anOspA polypeptide from Borrelia burgdorferi that includes residues 165,166 and 170, wherein the numbering corresponds to the numbering of SEQID NO: 7. The polynucleotide is altered such that residue 165 is changedto phenylalanine, residue 166 is changed to threonine or residue 170 ischanged to lysine or combination thereof. The altered polynucleotide isexpressed, thereby generating an altered Borrelia burgdorferi OspApolypeptide with reduced cross-reactivity with the hLFA-1 moleculecompared to the corresponding unaltered Borrelia burgdorferi OspApolypeptide. In another embodiment, an altered OspA polypeptide havingreduced cross-reactivity to hLFA-1 while retaining the ability to bebound by LA-2 is generated. In that embodiment, for example, residue 130is methionine, residue 160 is tyrosine, residue 165 is phenylalanine,residue 166 is threonine, residue 170 is lysine and residue 189 ismethionine. Polynucleotides encoding Borrelia burgdorferi OspApolypeptides can be selected as described herein.

[0101] In one embodiment, the altered OspA polypeptide includes theminimal sequence that includes the positions of the alterations. Forexample, the altered polypeptide can comprise OspA from about residue139 to about residue 189, wherein the numbering corresponds to SEQ IDNO: 7. In another embodiment, the altered polypeptide can comprise OspAfrom about residue 165 to about residue 170, wherein the numberingcorresponds to SEQ ID NO: 7. The altered OspA polypeptides of thepresent invention also include larger fragments of OspA. For example,the altered OspA polypeptides include, but are not limited to, alteredOspA polypeptides which comprise OspA from about residue 160 to aboutresidue 170, OspA from about residue 150 to 180, OspA from about residue131 to 273 or OspA from about residue 17 to 273. Methods of generatingand expressing varying-sized fragments of OspA which incorporate one ormore of the alterations described herein are described below and arewell known to those of skill in the art.

[0102] As described herein, the OspA sequence used to generate thealtered OspA polypeptide can itself be a chimeric OspA polypeptide,having two or more segments derived from OspA proteins from differentgenospecies or strains of Borrelia. The size of the altered OspApolypeptide can vary depending on the method used to generate thealtered polypeptide and/or the purpose for which it is generated, andsuch altered OspA chimeric polypeptides can include fragments of OspA.The altered polypeptide can be part of a larger polypeptide, includingadditional OspA sequences on the N-terminus, the C-terminus or bothtermini. A fragment of OspA protein can encompass polypeptides that areonly a part of the full-length OspA protein. Such OspA fragmentstypically include at least one of the altered residues described hereinand possess at least some of the antigenicity of wild type OspA. OspAfragments can be produced by amino and/or carboxyl terminal deletions,as well as internal deletions. Fragments can also be produced byenzymatic digestion. Such modified OspA molecules can be tested forantigenic activity as described herein or using methods known in theart.

[0103] In some embodiments, the altered OspA polypeptide can be part ofa cocktail with one or more other proteins, such as other Borreliaburgdorferi polypeptides, including but not limited to, OspA, OspB,OspC, OspD, p93 and p41. In other embodiments, the altered OspApolypeptide can be part of a larger molecule, such as a chimericpolypeptide, for example, as described in U.S. Pat. No. 6,248,562 andU.S. Patent Application entitled “Recombinant Constructs of Borreliaburgdorferi”, by Luft et al, filed on Aug. 7, 2001. Such largerpolypeptides can include amino acid sequences from other proteinsincluding but not limited to, other Borrelia burgdorferi proteins and/orother proteins useful in generating fusion proteins for vaccine and/orimmunodiagnostic methods. Additional components, for example, labels (aradioisotope, an epitope label (tag)(e.g., a hemagglutinin (HA) epitope,a hexahistidine tag), an affinity label (e.g., biotin, avidin), a spinlabel, an enzyme label, a fluorescent group, a chemiluminescent group)can be incorporated into the altered OspA polypeptides of the inventionto assist in the isolation and/or purification of the polypeptide. Forexample, a hexahistidine tag would permit ready purification by nickelchromatography. These and other components can also be incorporated inthe altered OspA polypeptides of the invention in order to extend thehalf life of the polypeptides. Methods of incorporating such componentsinto the polypeptides of the invention are well known to those of skillin the art.

[0104] In one embodiment, the altered OspA polypeptide of the inventionis a chimeric polypeptide. In a particular embodiment, the altered OspApolypeptide comprises the following: a) an amino acid sequence of afirst OspA polypeptide from about residue 1 to about residue 164 from afirst strain of Borrelia burgdorferi; b) an amino acid sequence of asecond OspA polypeptide from about residue 165 to about residue 179 froma second strain of Borrelia burgdorferi, wherein said second strain is adifferent strain from said first strain; c) an amino acid sequence of athird OspA polypeptide from about residue 180 to about residue 216 froma third strain of Borrelia burgdorferi, wherein said third strain is adifferent strain from said second strain; d) an amino acid sequence of afourth OspA polypeptide from about residue 217 to about residue 273 froma fourth strain of Borrelia burgdorferi, wherein said fourth strain is adifferent strain from said third strain; wherein the sequence includesat least one alteration selected from the group consisting of: residue139 being methionine, residue 160 being tyrosine, residue 189 beingmethionine and combinations thereof, wherein the numbering correspondsto the numbering of SEQ ID NO: 7.

[0105] Polypeptides described herein can be isolated fromnaturally-occurring sources, chemically synthesized or recombinantlyproduced. The altered OspA polypeptides of the present invention can bederived from naturally-occurring OspA molecules or from nucleic acidswhich encode such molecules. The OspA polypeptides of the presentinvention can comprise fragments, derivatives, analogs, variants andmutants of an OspA protein (modified OspA) and/or can be fragmented,derivatized or otherwise altered after having the alterations describedherein inserted (also referred to as modified OspA). Such modified OspAmolecules possess at least some OspA antigenic activity. According tothe invention, the amino acid sequence of the altered OspA polypeptidesof the invention can be that of a naturally-occurring protein or cancomprise additional modifications. Such additional modifications includeconservative and/or non-conservative amino acid substitutions, additionsof one or more amino acids, and/or deletions of one or more amino acids.Such additional modifications should also preserve at least someactivity of the encoded protein or polypeptide. For example, the furthermodified polypeptide or protein should have similar or improvedconformational stability, similar or improved immunoprotective activityor reduced cross-reactivity to hLFA-1, as compared to the correspondingaltered OspA polypeptide (i.e., the OspA polypeptide comprising one ormore of the alterations described herein but not comprising the furthermodification(s)).

[0106] For example, the further modification(s) preferably preserve thethree-dimensional configuration of an antibody binding site of thenative protein such as the LA-2 binding site. The presence or absence ofbiological activity or activities can be determined by variousfunctional assays as described herein or using methods that are known inthe art, e.g., recognition using an ELISA assay, elicitation of animmune response (e.g., an immunoprotective response) in an animal.Appropriate amino acid alterations that fall within the scope of theinvention can be made on the basis of several criteria, includinghydrophobicity, basic or acidic character, charge, polarity, size, thepresence or absence of a functional group (e.g., —SH or a glycosylationsite), and aromatic character, provided that the resulting molecule hasat least one of the alterations described herein and maintains increasedconformational stability and/or reduced cross-reactivity to hLFA-1.Assignment of various amino acids to similar groups based on theproperties above will be readily apparent to the skilled artisan;further appropriate amino acid changes can also be found in Bowie(Science, 247:1306-1310(1990)).

[0107] “Variants” and “mutants” of OspA can be produced using in vitroand/or in vivo techniques well-known to those of skill in the art, forexample, site-specific mutagenesis, and oligonucleotide mutagenesis.Manipulations of the OspA polypeptide sequence can be made at theprotein level as well. Chemical modifications can be carried out usingknown techniques including but not limited to, specific chemicalcleavage using cyanogen bromide, trypsin and/or papain. OspA can also bestructurally modified and/or denatured, for example, using heat. Ingeneral, mutations can be conservative or non-conservative amino acidsubstitutions, amino acid insertions or amino acid deletions.

[0108] For example, a nucleic acid (e.g., DNA) encoding a modified OspApolypeptide can be prepared by site-directed mutagenesis of the nucleicacid (e.g., DNA) that encodes a wild type OspA. Site-directed(site-specific) mutagenesis allows the production of OspA variantsthrough the use of specific oligonucleotide sequences that encode theDNA sequence of the desired mutation (e.g., alteration, deletion,insertion), as well as a sufficient number of adjacent nucleotides, toprovide a primer sequence of sufficient size and sequence complexity toform a stable duplex on both sides of the desired mutation. Typically, aprimer of about 20 to 25 nucleotides in length is preferred, with about5 to 10 complementary residues on both sides of the mutation of thesequence being altered. In general, the techniques of site-specificmutagenesis are well known in the art, as exemplified by publicationssuch as Edelman et al., DNA, 2:183, 1983. For example, a site-specificmutagenesis technique can employ a phage vector that exists in both asingle-stranded and double-stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage, forexample, as disclosed by Messing et al., Third Cleveland Symposium onMacromolecules and Recombinant DNA, A. Walton, ed., Elsevier, Amsterdam,1981. This and other phage vectors are commercially available and theiruse is well known to those skilled in the art. A versatile and efficientprocedure for the construction of oligonucleotide directed site-specificmutations in DNA fragments using M13-derived vectors was published byZoller, M. J. and Smith, M., Nucleic Acids Res., 10:6487-6500, 1982. Inaddition, plasmid vectors that contain a single-stranded phage origin ofreplication can be employed to obtain single-stranded DNA (see forexample, Veira et al., Meth Enzymol., 153:3, (1987)).

[0109] Alternatively, nucleotide substitutions can be introduced bysynthesizing the appropriate DNA fragment in vitro, and amplifying itusing PCR procedures known in the art.

[0110] In general, site-specific mutagenesis can be performed by firstobtaining a single-stranded vector that includes within its sequence aDNA sequence that encodes the relevant protein. An oligonucleotideprimer bearing the desired mutated sequence is prepared, generallysynthetically, for example, by the method of Crea et al., Proc Natl AcadSci USA., 75:5765, 1978. This primer can then be annealed with thesingle-stranded protein sequence-containing vector, and subjected to DNApolymerizing enzymes, e.g., E. coli polymerase I Klenow fragment, tocomplete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector can then be used to transform appropriate hostcells such as JM 101 cells, and clones can be selected that includerecombinant vectors bearing the mutated sequence arrangement.Thereafter, the mutated region can be removed and placed in anappropriate expression vector for protein production.

[0111] The PCR technique can be used in creating amino acid sequencevariants of OspA. When small amounts of template DNA are used asstarting material in a PCR, primers that differ slightly in sequencefrom the corresponding region in a template DNA can be used to generaterelatively large quantities of a specific DNA fragment that differs fromthe template sequence only at the positions where the primers differfrom the template. For introduction of a mutation into a plasmid DNA,one of the primers can be designed to overlap the position of themutation and to contain the mutation; the sequence of the other primeris preferably identical to a stretch of sequence of the opposite strandof the plasmid, but this sequence can be located anywhere along theplasmid DNA. It is preferred, however, that the sequence of the secondprimer be located within 500 nucleotides from that of the first, suchthat in the end the entire amplified region of DNA bounded by theprimers can be easily sequenced. PCR amplification using a primer pairlike the one just described results in a population of DNA fragmentsthat differ at the end position of the mutation specified by the primer.

[0112] The DNA fragments produced bearing the desired mutation can beused to replace the corresponding region in the plasmid that served asPCR template using standard DNA technology. Mutations at separatepositions can be introduced simultaneously by either using a mutantsecond primer or performing a second PCR with different mutant primersand ligating the two resulting PCR fragments simultaneously to thevector fragment in a three (or more) part ligation.

[0113] An additional method for preparing variants, cassettemutagenesis, is based on the technique described by Wells et al., Gene,34:315, 1985. The starting material can be the plasmid (or vector)comprising the OspA DNA to be mutated. The codon(s) within the OspA tobe mutated are identified. There must be unique restriction endonucleasesites on each side of the identified mutation site(s). If suchrestriction sites do not exist, they can be generated using theabove-described oligonucleotide-mediated mutagenesis method to introducethem at appropriate locations in the OspA DNA or they can be generatedusing PCR and the desired primers as described in the Exemplification.After the restriction sites have been introduced into the plasmid, theplasmid is cut at these sites to linearize it. A double strandedoligonucleotide encoding the sequence of the DNA between the restrictionsites but containing the desired mutation(s) is synthesized usingstandard procedures. The two strands are synthesized separately and thenhybridized together using standard techniques. This double-strandedoligonucleotide is referred to as the cassette. This cassette isdesigned to have 3′ and 5′ ends that are compatible with the ends of thelinearized plasmid, such that it can be directly ligated to the plasmid.The plasmid now contains the mutated OspA DNA sequence, and can besubcloned and/or expressed to produce the modified OspA polypeptide orprotein.

[0114] The OspA encoding nucleic acid molecules (e.g., polynucleotides)of the present invention have at least one of the alterations describedherein and generally hybridize under high stringency hybridizationconditions to a polynucleotide-encoding OspA nucleic acid or fragmentthereof from a sensu stricto strain of Borrelia burgdorferi, e.g., SEQID NO: 7. In one embodiment, the OspA-encoding nucleic acid molecules(e.g., polynucleotides) of the present invention hybridize under highstringency hybridization conditions to a polynucleotide encoding OspA orfragment thereof from Borrelia afzelii, e.g., SEQ ID NO: 10. In anotherembodiment, the OspA-encoding nucleic acid molecules (e.g.,polynucleotides) of the present invention hybridize under highstringency hybridization conditions to a polynucleotide encoding OspA orfragment thereof from Borrelia garinii, e.g., SEQ ID NO: 8. Thus, thepolynucleotides and polypeptides of the present invention includemodified versions of OspA as described herein.

[0115] Appropriate selective stringency conditions are known to thoseskilled in the art or can be found in standard texts such as CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. For example, stringent hybridization conditions include asodium ion concentration of no more than 1 M and a temperature of atleast 25° C. In one embodiment, conditions of 5×SSPE (750 mM NaCl, 50 mMNaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30° C., orequivalent conditions, are suitable for specific hybridization.Equivalent conditions can be determined by varying one or more of theparameters, as is known in the art, while maintaining a similar degreeof identity or similarity between the target nucleic acid molecule andthe primer or probe used. Hybridizable nucleic acid molecules are usefulas probes and primers for diagnostic applications.

[0116] Accordingly, the invention pertains to nucleic acid moleculeswhich have a substantial identity with the nucleic acid moleculesencoding the altered OspA polypeptides described herein wherein thenucleic acid encodes for one or more of the alterations describedherein; particularly preferred are nucleic acid molecules which have atleast about 90%, more preferably at least about 95% and most preferablyat least about 98% identity with nucleic acid molecules describedherein, wherein the nucleic acid encodes for at least one of thealterations described herein. Sequence identity can be determined usingpublically or commercially available sequence alignment algorithms,using, for example, default parameters.

[0117] Thus, DNA molecules which comprise a sequence which is differentfrom the naturally-occurring nucleic acid molecule but which, due to thedegeneracy of the genetic code, encode the same protein or polypeptideare encompassed by the present invention. The invention also encompassesvariations of the nucleic acid molecules of the invention, such as thoseencoding portions, analogues or derivatives of the encoded protein orpolypeptide. Such variations can be naturally-occurring, such as in thecase of allelic variation, or non-naturally-occurring, such as thoseinduced by various mutagens and mutagenic processes, so long as thenucleic acid molecule encodes at least one of the alterations describedherein and the encoded protein has either increased conformationalstability and/or decreased cross-reactivity to hLFA-1 (as compared tothe corresponding unaltered protein) that is conferred by thealterations described herein. Intended variations include, but are notlimited to, addition, deletion and/or substitution of one or morenucleotides which can result in conservative or non-conservative aminoacid changes, including additions and deletions. Preferably, suchnucleotide or amino acid variations are silent; that is, they do notalter one or more characteristics or activity of the encoded alteredOspA protein or polypeptide. As used herein, activities of the encodedprotein or polypeptide include, but are not limited to, bindingfunction, antigenic function and conformational stability.

[0118] The invention also provides expression vectors containing anucleic acid sequence described herein, operably linked to at least oneregulatory sequence. Many such vectors are commercially available, andother suitable vectors can be readily prepared by the skilled artisan.“Operably linked” is intended to mean that the nucleic acid molecule islinked to a regulatory sequence in a manner which allows expression ofthe nucleic acid sequence. Regulatory sequences are art-recognized andare selected to produce the encoded polypeptide or protein. Accordingly,the term “regulatory sequence” includes promoters, enhancers, and otherexpression control elements which are described in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). For example, the native regulatory sequences orregulatory sequences native to the transformed host cell can beemployed. It should be understood that the design of the expressionvector may depend on such factors as the choice of the host cell to betransformed and/or the type of protein desired to be expressed. Forinstance, the polypeptides of the present invention can be produced byligating the cloned gene, or a portion thereof, into a vector suitablefor expression in either prokaryotic cells, eukaryotic cells or both(see, for example, Broach, et al., Experimental Manipulation of GeneExpression, ed. M. Inouye (Academic Press, 1983) p. 83; MolecularCloning: A Laboratory Manual, 2nd Ed., ed. Sambrook et al., (Cold SpringHarbor Laboratory Press, 1989) Chapters 16 and 17). Typically,expression constructs will contain one or more selectable markers,including but not limited to, the gene that encodes dihydrofolatereductase and the genes that confer resistance to neomycin,tetracycline, ampicillin, chloramphenicol, kanamycin or streptomycin.

[0119] Prokaryotic and eukaryotic host cells transfected by thedescribed vectors are also provided by this invention. For instance,cells which can be transfected with the vectors of the present inventioninclude, but are not limited to, bacterial cells such as E. coli (e.g.,E. coli K12, BL21, DH5αstrains), Streptomyces, Pseudomonas, Serratiamarcescens and Salmonella typhimurium, insect cells (baculovirus)including Drosophila, fungal cells, such as yeast cells, plant cells andmammalian cells, such as thymocytes, Chinese hamster ovary cells (CHO)and COS cells.

[0120] Thus, a nucleic acid molecule comprising, for example SEQ ID NO:6 with at least one of the specific alterations described herein, or anucleic acid molecule which encodes, for example SEQ ID NO: 7 with atleast one of the specific alterations described herein, can be used toproduce a recombinant form of the protein via microbial or eukaryoticcellular processes. Ligating the nucleic acid molecule (e.g.,polynucleotide) into a gene construct, such as an expression vector, andtransforming or transfecting into hosts, either eukaryotic (yeast,avian, insect, plant or mammalian) or prokaryotic (bacterial cells), arestandard procedures used in producing other well known proteins. Similarprocedures, or modifications thereof, can be employed to preparerecombinant proteins according to the present invention by microbialmeans or tissue-culture technology. Accordingly, the invention pertainsto the production of encoded proteins or polypeptides by recombinanttechnology.

[0121] The proteins and polypeptides of the present invention can beisolated or purified (e.g., to homogeneity) from recombinant cellculture by a variety of processes. These include, but are not limitedto, anion or cation exchange chromatography, ethanol precipitation,affinity chromatography and high performance liquid chromatography(HPLC). The particular method used will depend upon the properties ofthe polypeptide and the selection of the host cell; appropriate methodswill be readily apparent to those skilled in the art.

[0122] The present invention also pertains to pharmaceuticalcompositions comprising polypeptides and other compounds describedherein. For instance, a polypeptide or protein of the present inventioncan be formulated with a physiologically acceptable medium to prepare apharmaceutical composition. The particular physiological medium mayinclude, but is not limited to, water, buffered saline, polyols (e.g.,glycerol, propylene glycol, liquid polyethylene glycol) and dextrosesolutions. The optimum concentration of the active ingredient(s) in thechosen medium can be determined empirically, according to well knownprocedures, and will depend on the ultimate pharmaceutical formulationdesired. Methods of introduction of exogenous polypeptides at the siteof treatment include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, oral andintranasal. Other suitable methods of introduction can also include genetherapy, rechargeable or biodegradable devices and slow releasepolymeric devices. The pharmaceutical compositions of this invention canalso be administered as part of a combinatorial therapy with otheragents.

[0123] The altered OspA proteins described herein can be produced sothat they are highly soluble, hyper-produced in E. coli, andnon-lipidated. In addition, the altered OspA proteins can be designed tobegin or end in a suitable affinity tag (e.g., a His-tag) to facilitatepurification. The recombinant proteins described herein have beenconstructed to maintain high levels of antigenicity and improvedconformational stability.

[0124] The altered OspA proteins of the current invention areadvantageous in that they retain at least some specific reactivity tomonoclonal and/or polyclonal antibodies against wild-type Borreliaproteins, are immunogenic, and inhibit the growth or induce lysis ofBorrelia in vitro. The proteins are particularly useful inimmunodiagnostic assays. For example, proteins of the present inventioncan be used as reagents in assays to detect the presence of antibodiesto native Borrelia in potentially infected individuals. These proteinscan also be used as immunodiagnostic reagents, such as in dot blots,Western blots, enzyme-linked immunosorbent assays, or agglutinationassays. The altered OspA proteins of the present invention can beproduced by known techniques, such as by recombinant methodology,polymerase chain reaction, or mutagenesis.

[0125] Furthermore, the proteins of the current invention are useful asvaccine immunogens against Borrelia infection. One or more of thealtered proteins can be combined with a physiologically acceptablecarrier and administered to a vertebrate animal through standard methods(e.g., intravenously or intramuscularly, for example).

[0126] The altered forms of the OspA proteins described herein werebioengineered such that at least one immunoprotective domain of theprotein was maintained. As described herein, antigenic refers to theability of a compound to bind products of an immune response, such asantibodies, T-cell receptors or both. Such responses can be measuredusing standard antibody detection assays, such as ELISA or standardT-cell activation assays. In a preferred embodiment, the altered formsof OspA described herein elicit an immunoprotective response, forexample, by eliciting antibodies that recognize the LA-2 epitope.

[0127] It is understood that the nucleic acids that encode thepolypeptides that comprise the altered OspA protein can include extranucleotides or fewer nucleotides in order to simplify the constructionof the gene encoding the chimeric polypeptide, e.g., to allow for theuse of convenient restriction endonuclease sites, or to allow theligation of the gene fragments such that a contiguous coding region iscreated. Based on the guidance provided herein, one of ordinary skill inthe art would readily be able to add or remove nucleotides from thetermini of the gene fragments encoding the polypeptides of the OspAprotein in order to generate the altered OspA proteins of the presentinvention with no experimentation or using only routine experimentation.The altered OspA polypeptides of the present invention can be lipidatedor non-lipidated.

[0128] To test the antigenicity of the altered OspA polypeptides, micecan be immunized with OspA polypeptides or proteins containing thepolypeptide sequences in aluminum hydroxide. Mice are then bled andtested for antibody responses against OspA derived from various strainsof Borrelia. In additional experiments, these immunized mice can bechallenged with ticks infected with Borrelia burgdorferi andtransmission of infection can be assessed as described in theExemplification which use OspA, OspC and OspC/OspA chimeric molecules.The results of such a tick challenge reveals whether the animal hasdeveloped a protective immune response. For example, an immunized animalthat does not seroconvert in response to subsequent tick challenge haslikely generated an immunoprotective response to the immunization.

[0129] The immunogenic compositions of the present invention can be usedto immunize animals including humans. Immunization is understood toelicit specific immunogenic responses, preferably immunoprotectiveresponses, as described above. As described herein, an immunogenicresponse includes responses that result in at least some level of immuneresponse in the treated animal, where the animal was previously treatedwith a composition comprising at least one altered OspA polypeptide ofthe present invention.

[0130] Immunity, as described herein, is understood to mean the abilityof the treated animal to resist infection, to resist systemic infection,or to overcome infection such as systemic infection more easily or morequickly when compared to non-immunized or non-treated individuals.Immunity can also include an improved ability of the treated individualto sustain an infection with reduced or no clinical symptoms of systemicinfection. The individual may be treated with the altered OspA proteinsof the present invention either proactively, e.g., once a year or,alternatively, after sustaining a tick bite.

[0131] In one embodiment, the altered OspA protein of the presentinvention, together with suitable excipients and/or adjuvants, isadministered to an animal such that the animal develops an immuneresponse to the OspA polypeptide of the composition. The pharmaceuticalcomposition can also be administered with other components suitable forin vitro and/or in vivo use. These additional components includebuffers, carrier proteins, adjuvants, preservatives and combinationsthereof. In a preferred embodiment, the individual generates animmunoprotective response, for example, by generating antibodies thatrecognize the LA-2 epitope.

[0132] The present invention is also drawn to a physiologicalcomposition comprising an altered OspA protein. The composition isuseful to administer to an animal in order to generate an immuneresponse or in the diagnostic methods described herein.

[0133] For use as a vaccine, the composition of the present inventioncan include suitable adjuvants, well known in the art, to enhanceimmunogenicity, potency or half-life of the chimeric proteins in thetreated animal. Adjuvants and their use are well known in the art (seefor example PCT Publication WO 96/40290, the entire teachings of whichare incorporated herein by reference). The composition can be preparedby known methods of preparing vaccines. For example, the altered OspApolypeptides described herein can be isolated and/or purified usingknown techniques, such as by size exclusion chromatography, ion exchangechromatography, affinity chromatography, preparative electrophoresis,selective precipitation or combinations thereof. The prepared proteinscan be mixed with suitable other reagents as described above, whereinthe protein is at a suitable concentration. The dosage of the proteinwill vary and depends upon the age, weight and/or physical condition ofthe animal to be treated. The optimal dosage can be determined byroutine optimization techniques, using suitable animal models.

[0134] The composition to be used as a vaccine can be administered byany suitable technique. In one embodiment, administration is byinjection, e.g., subcutaneous, intramuscular, intravenous, or intraperitoneal injection. In another embodiment, the composition isadministered to mucosa, e.g., by exposing nasal mucosa to nose dropscontaining the proteins of chimeric proteins of the present invention.In another embodiment, the immunogenic composition is administered byoral administration. In another embodiment of the present invention thechimeric proteins are administered by DNA immunization using nucleicacids encoding an altered OspA polypeptide.

[0135] The present invention is also drawn to a diagnostic kitcomprising the altered OspA polypeptides described herein. The kit alsoincludes reagents for detecting antibody-antigen complexes that areformed between the OspA protein and antibodies that are present in ansample, e.g., a user-supplied host sample.

[0136] The present invention is also drawn to methods of detecting animmune response to Lyme Disease-causing Borrelia in a host sample. Themethod comprises contacting a host sample with an OspA altered protein,such that anti-OspA antibodies, if present in said sample, bind to saidOspA protein. The quantity of antibodies that have bound said OspAprotein are measured, thereby detecting an immune response to Lymedisease-causing Borrelia.

EXEMPLIFICATION Example 1 Purification of Borrelia burgdorferi OuterSurface Protein A and Analysis of Antibody Binding Domains

[0137] This example details a method for the purification of largeamounts of native outer surface protein A (OspA) to homogeneity, anddescribes mapping of the antigenic specificities of several anti-OspAMAbs. OspA was purified to homogeneity by exploiting its resistance totrypsin digestion. Intrinsic labeling with ¹⁴C-palmitic acid confirmedthat OspA was lipidated, and partial digestion established lipidation atthe amino-terminal cysteine of the molecule.

[0138] The reactivity of seven anti-OspA murine monoclonal antibodies tonine different Borrelia isolates was ascertained by Western blotanalysis. The reactivity of the altered OspA polypeptides describedherein was tested using similar methods. Intact, lipidated ornon-lipidated OspA and altered OspA can also be tested using similarmethods. Purified OspA was fragmented by enzymatic or chemical cleavage,and the monoclonal antibodies were able to define four distinctimmunogenic domains (see FIG. 1). Domain 3, which included residues190-220 of OspA, was reactive with protective antibodies known toagglutinate the organism in vitro, and included distinct specificities,some of which were not restricted to a genotype of B. burgdorferi.

[0139] A. Purification of Native OspA

[0140] Detergent solubilization of B. burgdorferi strips the outersurface proteins and yields partially-purified preparations containingboth OspA and outer surface protein B (OspB) (Barbour, A. G. et al.,Infect. Immun., 52(5):549-554 (1986); Coleman, J. L. et al., J. Infect.Dis., 155 (4):756-765 (1987); Cunningham, T. M. et al., Ann. NY Acad.Sci., 539:376-378 (1988); Brandt, M. E. et al., Infect. Immun., 58:983-991 (1990); Sambri, V. and R. Cevenini, Microbiol., 14:307-314(1991)). Although both OspA and OspB are sensitive to proteinase Kdigestion, in contrast to OspB, OspA is resistant to cleavage by trypsin(Dunn, J. et al., Prot. Exp. Purif., 1:159-168 (1990); Barbour, A. G. etal., Infect. Immun., 45:94-100 (1984)). The relative insensitivity totrypsin is surprising in view of the fact that OspA has a high (16% forB31) lysine content, and may relate to the relative configuration ofOspA and B in the outer membrane.

[0141] Intrinsic Radiolabeling of Borrelia

[0142] Labeling for lipoproteins was performed as described by Brandt etal. (Brandt et al., Infect. Immun., 58:983-991 (1990)). ¹⁴C-palmiticacid (ICN, Irvine, Calif.) was added to the BSK II media to a finalconcentration of 0.5 μCi per milliliter (ml). Organisms were cultured at34° C. in this medium until a density of 10⁸ cells per ml was achieved.

[0143] Purification of OspA Protein from Borrelia Strain B31

[0144]Borrelia burgdorferi, either ¹⁴C-palmitic acid-labeled orunlabeled, were harvested and washed as described (Brandt, M. E. et al.,Infect. Immun., 58:983-991 (1990)). Whole organisms were trypsinizedaccording to the protocol of Barbour et al., (Infect. Immun., 45:94-100(1984)) with some modifications. The pellet was suspended in phosphatebuffered saline (PBS, 10 mM, pH 7.2), containing 0.8%tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated trypsin (Sigma,St. Louis, Mo.), the latter at a ratio of 1 μg per 10⁸ cells. Reactionwas carried out at 25° C. for 1 hour, following which the cells werecentrifuged. The pellet was washed in PBS with 100 μg/mlphenylmethylsulfonyl fluoride (PMSF). Triton X-114 partitioning of thepellet was carried out as described by Brandt et al., (Brandt et al.,Infect. Immun., 58:983-991 (1990)). Following trypsin treatment, cellswere resuspended in ice-cold 2% (v/v) Triton X-114 in PBS at 10⁹ cellsper ml. The suspension was rotated overnight at 4° C., and the insolublefraction removed as a pellet after centrifugation at 10,000×g for 15minutes at 4° C. The supernatant (soluble fraction) was incubated at 37°C. for 15 minutes and centrifuged at room temperature at 1000×g for 15minutes to separate the aqueous and detergent phases. The aqueous phasewas decanted, and ice cold PBS added to the lower Triton phase, mixed,warmed to 37° C., and again centrifuged at 1000×g for 15 minutes.Washing was repeated twice more. Finally, detergent was removed from thepreparation using a spin column of Bio-beads SM2 (BioRad, Melville,N.Y.) as described (Holloway, P. W., Anal. Biochem., 53:304-308 (1973)).

[0145] Ion exchange chromatography was carried out as described by Dunnet al., (Dunn et al., Prot. Exp. Purif., 1:159-168 (1990)) with minormodifications. Crude OspA was dissolved in buffer A (1% Triton X-100, 10mM phosphate buffer (pH 5.0)) and loaded onto a SP Sepharose resin(Pharmacia, Piscataway, N.J.), pre-equilibrated with buffer A at 25° C.After washing the column with 10 bed-volumes of buffer A, the bound OspAwas eluted with buffer B (1% Triton X-100, 10 mM phosphate buffer (pH8.0)). OspA fractions were detected by protein assay using the BCAmethod (Pierce, Rockford, Ill.), or as radioactivity when intrinsicallylabeled material was fractionated. Triton X-100 was removed using a spincolumn of Bio-beads SM2.

[0146] This method purifies OspA from an outer surface membranepreparation. In the absence of trypsin-treatment, OspA and B were themajor components of the soluble fraction obtained after Tritonpartitioning of strain B31. In contrast, when Triton extraction wascarried out after trypsin-treatment, the OspB band is not seen. Furtherpurification of OspA-B31 on a SP Sepharose column resulted in a singleband by SDS-PAGE. The yield following removal of detergent wasapproximately 2 mg per liter of culture. This method of purification ofOspA, as described herein for strain B31, can be used for other isolatesof Borrelia as well. For strains such as strain K48, which lack OspB,trypsin treatment can be omitted.

[0147] Lipidation Site of OspA-B31

[0148]¹⁴C-palmitic acid labeled OspA from strain B31 was purified asdescribed above and partially digested with endoproteinase Asp-N.Following digestion, a new band of lower molecular weight was apparentby SDS-PAGE, found by direct amino-terminal sequencing to begin atAsp₂₅. This band had no trace of radioactivity by autoradiography. OspAand B contain a signal sequence (L-X-Y-C) similar to the consensusdescribed for lipoproteins of E. coli, and it has been predicted thatthe lipidation site of OspA and B should be the amino-terminal cysteine(Brandt, M. E. et al., Infect. Immun, 58:983-991 (1990)). The resultspresented herein support this prediction.

[0149] B. Comparison of OspA Antibody Binding Regions in Nine Strains ofBorrelia burgdorferi

[0150] The availability of the amino acid sequenced for OspA from anumber of different isolates, combined with peptide mapping and Westernblot analysis, permitted the identification of the antigenic domainsrecognized by monoclonal antibodies (MAbs) and allowed inference of thekey amino acid residues responsible for specific antibody reactivity.

[0151] Strains of Borrelia burgdorferi

[0152] Nine strains of Borrelia, including seven European strains andtwo North American strains, were used in this study of antibody bindingdomains of several proteins. Information concerning the strains issummarized in Table I, below. TABLE I Representative Borrelia StrainsStrain Location and Source Reference for Strain K48 Czechoslovakia, noneIxodes ricinus PGau Germany, human ACA Wilske, B. et al., J. Clin.Microbiol. 32:340-350 (1993) DK29 Denmark, human EM Wilske, B. et al.Pko Germany, human EM Wilske, B. et al. PTrob Germany, human skinWilske, B. et al. Ip3 Khabarovsk, Russia, Asbrink, E. et al., Acta Derm.I. persulcatus Venereol., 64:506-512 (1984) Ip90 Khabarovsk, Russia,Asbrink, E. et al. persulcatus 25015 Milibrook, NY, Barbour, A. G. etal., Curr. Microbiol., I. persulcatus 8:123-126 (1983) B31 ShelterIsland, NY, Luft, B. J. et al., Infect. Immun, 60: I. scapularis4309-4321 (1992); ATCC 35210 PKa1 Germany, human CSF Wilske, B. et al.ZS7 Freiburg, Germany, Wallich, R. et al., Nucl. Acids Res, I. ricinus17:8864 (1989) N40 Westchester Co., NY Fikrig, B. et al., Science,250:553-556 (1990) PHei Germany, human CSF Wilske, B. et al. ACAISweden, human ACA Luft, B. J. et al., FEMS Microbiol. Lett. 93:73-68(1992) PBo Germany, human CSF Wilske, B. et al.

[0153] Strains K48, PGau and DK29 were supplied by R. Johnson,University of Minnesota; Pko and pTrob were provided by B. Wilske and V.Preac-Mursic of the Pettenkhofer Institute, Munich, Germany; and Ip3 andIp90 were supplied by L. Mayer of the Center for Disease Control,Atlanta, Ga. The North American strains included strain 25015, providedby J. Anderson of the Connecticut Department of Agriculture; and strainB31 (ATCC 35210).

[0154] Monoclonal Antibodies

[0155] Seven monoclonal antibodies (MAbs) were utilized in this study.Five of the MAbs (12, 13, 15, 83 and 336) were produced from hybridomascloned and subcloned as previously described (Schubach, W. H., et al.,Infect. Immun., 59(6):1911-1915 (1991)). MAb H5332 (Barbour, A. G. etal., Infect. Immun., 41: 795-804 (1983)) was a gift from Drs. AlanBarbour, University of Texas, and MAb CIII.78 (Sears, J. E. et al., J.Immunol., 147(6):1995-2000 (1991)) was a gift from Richard A. Flavell,Yale University. MAbs 12 and 15 were raised against whole sonicated B3;MAb 336 was produced against whole PGau; and MAbs 13 and 83 were raisedto a truncated form of OspA cloned from the K48 strain and expressed inE. coli using the T7 RNA polymerase system (McGrath, B. C. et al.,Vaccines, Cold Spring Harbor Laboratory Press, Plainview, N.Y., pp.365-370 (1993)). All MAbs were typed as being Immunoglobulin G (IgG).

[0156] Methods of Protein Cleavage, Western Blotting and Amino-TerminalSequencing

[0157] Prediction of the various cleavage sites was achieved byknowledge of the primary amino acid sequence derived from the fullnucleotide sequences of OspA, many of which are currently available (seeTable II, below). Cleavage sites can also be predicted based on thepeptide sequence of OspA, which can be determined by standard techniquesafter isolation and purification of OspA by the method described above.Cleavage of several OspA isolates was conducted to determine thelocalization of monoclonal antibody binding of the proteins.

[0158] Hydroxylamine-HCl (HA), N-chlorosuccinimide (NCS), and cyanogenbromide cleavage of OspA followed the methods described by Bornstein(Biochem. 9 (12):2408-2421 (1970)), Shechter et al., (Biochem., 15(23):5071-5075 (1976)), and Gross (in Hirs, C. H. W. (ed): Methods inEnzymology, (N.Y. Acad. Press), 11:238-255 (1967)) respectively.Protease cleavage by endoproteinase, Asp-N (Boehringer Mannheim,Indianapolis, Ind.), was performed as described by Cleveland D. W. etal., (J. Biol. Chem., 252:1102-1106 (1977)). Ten micrograms of OspA wereused for each reaction. The ratio of enzyme to OspA was approximately 1to 10 (w/w).

[0159] Proteins and peptides generated by cleavage were separated bySDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, U. K.,Nature (London) 227:680-685 (1970)), and electroblotted onto immobilonPolyvinylidine Difluoride (PVDF) membranes (Ploskal, M. G. et al.,Biotechniques, 4:272-283 (1986)). They were detected by amido blackstaining or by immunostaining with murine MAbs, followed by alkalinephosphatase-conjugated goat anti-mouse IgG. Specific binding wasdetected using a 5-bromo-4-chloro-3-indolylphosphate (BCIP)/nitrobluetetrazolium (NBT) developer system (KPL Inc., Gathersburg, Md.).

[0160] In addition, amino-terminal amino acid sequence analysis wascarried out on several cleavage products, as described by Luft et al.,(Infect. Immun., 57:3637-3645 (1989)). Amido black stained bands wereexcised from PVDF blots and sequenced by Edman degradation using aBiosystems model 475A sequenator with model 120A PTH analyzer and model900A control/data analyzer.

[0161] Cleavage Products of Outer Surface Protein A Isolates

[0162] Purified OspA-B31, labeled with ¹⁴C-palmitic acid, was fragmentedwith hydroxylamine-HCl (HA) into two peptides, designated HA1 and HA2(data not shown). The HA1 band migrated at 27 kd and retained itsradioactivity, indicating that the peptide included the lipidation siteat the N-terminus of the molecule (data not shown). From the predictedcleavage point, HA1 should correspond to residues 1 to 251 of OspA-B31.HA2 had a MW of 21.6 kd by SDS-PAGE, with amino-terminal sequenceanalysis showing it to begin at Gly72, i.e. residues 72 to 273 ofOspA-B31. By contrast, HA cleaved OspA-K48 into three peptides,designated HA1, HA2, and HA3 with apparent MWs of 22 kd, 16 kd and 12kd, respectively. Amino-terminal sequencing showed HA1 to start atGly72, and HA3 at Gly142. HA2 was found to have a blockedamino-terminus, as was observed for the full-length OspA protein. HA1, 2and 3 of OspA-K48 were predicted to be residues 72-274, 1 to 141 and 142to 274, respectively.

[0163] N-Chlorosuccinimide (NCS) cleaves tryptophan (W), which is atresidue 216 of OspA-B31 or residue 217 of OspA-K48 (data not shown). NCScleaved OspA-B31 into 2 fragments, NCS1, with MW of 23 kd, residues1-216 of the protein, and NCS2 with a MW of 6.2 kd, residues 217 to 273(data not shown). Similarly, K48 OspA was divided into 2 pieces, NCS1residues 1-217, and NCS2 residues 218 to 274 (data not shown).

[0164] Cleavage of OspA by cyanogen bromide (CNBr) occurs at the carboxyside of methionine, residue 39. The major fragment, CNBr1, has a MW of25.7 kd, residues 39-274 by amino-terminal amino acid sequence analysis(data not shown). CNBr2 (about 4 kd) could not be visualized by amidoblack staining; instead, lightly stained bands of about 20 kd MW wereseen. These bands reacted with anti-OspA MAbs, and most likely weredegradation products due to cleavage by formic acid.

[0165] Determination of Antibody Binding Domains for Anti-OspAMonoclonal Antibodies

[0166] The cleavage products of OspA-B31 and OspA-K48 were analyzed byWestern blot to assess their ability to bind to the six different MAbs.Preliminary Western blot analysis of the cleavage products demonstratedthat strains K48 and DK29 have similar patterns of reactivity, as doIP3, PGau and Pko. The OspA of strain PTrob was immunologically distinctfrom the others, being recognized only by MAb 336. MAb 12 recognizedonly the two North American strains, B31 and 25015. When the isolateswere separated into genogroups, it was remarkable that all the MAbs,except MAb 12, crossed over to react with multiple genogroups.

[0167] MAb 12, specific for OspA-B31, bound to both HA1 and HA2 ofOspA-B31. However, cleavage of OspA-B31 by NCS at residue Trp216 createdfragments which did not react with MAb12, suggesting that the relevantdomain is near or is structurally dependent upon the integrity of thisresidue (data not shown). MAb 13 bound only to OspA-K48, and to peptidescontaining the amino-terminus of that molecule (e.g. HA2; NCS1). It didnot bind to CNBr1 residues 39 to 274. Thus the domain recognized byMAb13 is in the amino-terminal end of OspA-K48, near Met38.

[0168] MAb15 reacts with the OspA of both the B31 and K48 strains, andto peptides containing the N-terminus of OspA, such as HA1 of OspA-B31and NCS1, but not to peptides HA2 of OspA-B31 and HA1 of OspA-K48 (datanot shown). Both peptides include residue 72 to the C-terminus of themolecules. MAb15 bound to CNBr1 of OspA-K48, indicating the domain forthis antibody to be residues 39 to 72, specifically near Gly72 (data notshown).

[0169] MAb83 binds to OspA-K48, and to peptides containing theC-terminal portion of the molecule, such as HA1. They do not bind to HA2of OspA-K48, most likely because the C-terminus of HA2 of OspA-K48 endsat 141. Similar to MAb12 and OspA-B31, binding of MAbs 83 and CIII.78 iseliminated by cleavage of OspA at the tryptophan residue. Thus bindingof MAbs 12, 83 and CIII.78 to OspA depends on the structural integrityof the Trp₂₁₆ residue, which appears to be critical for antigenicity.Also apparent is that, although these MAbs bind to a common antigenicdomain, the precise epitopes which they recognize are distinct from oneanother given the varying degrees of cross-reactivity to these MAbsamong strains.

[0170] Although there is similar loss of binding activity of MAb336 withcleavage at Trp₂₁₆, this MAb does not bind to HA1 of OspA-B31,suggesting the domain for this antibody includes the carboxy-terminalend of the molecule, inclusive of residues 251 to 273. Low MW peptides,such as HA3 (10 kd) and NCS2 (6 kd), of OspA-K48 do not bind this MAb onWestern blots. In order to confirm this observation, we tested bindingof the 6 MAbs with a recombinant fusion construct p3A/EC that contains atrpE leader protein fused with residues 217 to 273 of OspA-B31(Schubach, W. H. et al., Infect. Immun., 59(6):1911-1915 (1991)). OnlyMAb336 reacted with this construct (data not shown). Peptides andantigenic domains localized by fragmentation of OspA are summarized inFIG. 1.

Example 2 Site-Directed Mutagenesis within Hypervariable Domains A.,(Residues 120-140), B., (Residues 150-180) and C., (Residues 200-216 or217)

[0171] Site-directed mutagenesis was performed to convert residueswithin the 204-219 domain of the recombinant B31 OspA to the analogousresidues of a European OspA variant, K48. In the region of OspA betweenresidues 204 and 219, there are seven amino acid differences betweenOspA-B31 and OspA-K48. Three oligonucleotides were generated, eachcontaining nucleotide changes which would incorporate K48 amino acids attheir analogous positions in the B31 OspA protein. The oligonucleotidesused to create the site-directed mutants were:

[0172] 5′-CTTAATGACTCTGACACTAGTGC-3′ (#613, which converts threonine atposition 204 to serine, and serine at 206 to threonine (Thr204-Ser,Thr206-Ser)) (SEQ ID NO: 1); 5′-GCTACTAAAAAAACCGGGAAATGGAATTCA-3′ (#625,which converts alanine at 214 to glycine, and alanine at 215 to lysine(Ala214-Gly, Ala215-Lys)) (SEQ ID NO: 2); and

[0173] 5′-GCAGCTTGGGATTCAAAAACATCCACTTTAACA-3′ (#640, which convertsasparagine at 217 to aspartate, and glycine at 219 to lysine(Asn217-Asp, Gly219-Lys)) (SEQ ID NO: 3).

[0174] Site-directed mutagenesis was carried out by performingmutagenesis with pairs of the above oligonucleotides. Threesite-directed mutants were created, each with two changes: OspA 613(Thr204-Ser, Thr206-Ser), OspA 625 (Ala214-Gly, Ala215-Lys), and 640(Asn217-Asp, Gly219-Lys). There were also two proteins with fourchanges: OspA 613/625 (Thr204-Ser, Thr206-Ser, Ala214-Gly, Ala215-Lys)and OspA 613/640 (Thr204-Ser, Thr206-Ser, Asn217-Asp, Gly219-Lys).

[0175] Specificity of Antibody Binding to Epitopes of the Non-mutatedHypervariable Region

[0176] Monoclonal antibodies that agglutinate spirochetes, includingseveral which are neutralizing in vitro, recognize epitopes that map tothe hypervariable region around Trp216 (Barbour, A. G. et al., Infect.and Immun., 41:759 (1983); Schubach, W. H. et al., Infect. and Immun.,59:1911 (1991)). Western Blot analysis demonstrated that chemicalcleavage of OspA from the B31 strain at Trp 216 abolishes reactivity ofthe protein with the agglutinating MAB 105, a monoclonal raised againstB31 spirochetes. The reagent, n-chlorosuccinimide (NCS), cleaves OspA atthe Trp 216, forming a 23.2 kd fragment and a 6.2 kd peptide which isnot retained on the Imobilon-P membrane after transfer. The uncleavedmaterial binds MAb 105; however, the 23.2 kd fragment is unreactive.Similar Western blots with a TrpE-OspA fusion protein containing thecarboxy-terminal portion of the OspA protein demonstrated that the small6.2 kd piece also fails to bind MAb 105 (Schubach, W. H. et al., Infect.and Immun., 59:1911 (1991)).

[0177] Monoclonal antibodies H5332 and H3TS (Barbour, A. G. et al.,Infect. and Immun., 41:759 (1983)) have been shown by immunofluorescenceto decorate the surface of fixed spirochetes (Wilske, B. et al., WorldJ. Microbiol., 7:130 (1991)). These monoclonal antibodies also inhibitthe growth of the organism in culture. Epitope mapping with fusionproteins has confirmed that the epitopes which bind these MAbs areconformationally determined and reside in the carboxy half of theprotein. MAb H5332 is cross-reactive among all of the known phylogeneticgroups, whereas MAb H3TS and MAb 105 seem to be specific to the B31strain to which they were raised. Like MAb 105, the reactivities ofH5332 and H3TS to OspA are abrogated by fragmentation of the protein atTrp216. MAb 336 was raised to whole spirochetes of the strain PGau. Itcross-reacts to OspA from group 1 (the group to which B31 belongs) butnot to group 2 (of which K48 is a member). Previous studies using fusionproteins and chemical cleavage have indicated that this antibodyrecognizes a domain of OspA in the region between residues 217 and 273.All of these MAbs agglutinate the B31 spirochete.

[0178] Western Blot Analysis of Antibody Binding to MutatedHypervariable Regions

[0179] MAbs were used for Western Blot analysis of the site-directedOspA mutants induced in E. coli using the T7 expression system (Dunn, J.J. et al., Protein Expression and Purification, 1:159 (1990)). E. colicells carrying pET9c plasmids having a site-directed OspA mutant insertwere induced at mid-log phase growth with IPTG for four hours at 37° C.Cell lysates were made by boiling an aliquot of the induced cultures inSDS gel loading dye, and this material was then loaded onto a 12% SDSgel (BioRad mini-Protean II), and subjected to electrophoresis. Theproteins were then transferred to Imobilon-P membranes (Millipore) 70V,2 hour at 4° C. using the BioRad mini transfer system. Western analysiswas carried out as described by Schubach et al., (Infect. Immun.,59:1911 (1991)).

[0180] Western Blot analysis indicated that only the 625 mutant(Ala214-Gly and Ala215-Lys) retained binding to the agglutinatingmonoclonal H3TS. However, the 613/625 mutant which has additionalalterations to the amino terminus of Trp216 (Ser204-Thr and Thr206-Ser)did not bind this monoclonal. Both 640 and 613/640 OspAs which have theAsn217-Asp and Gly219-Lys changes on the carboxy-terminal side of Trp216also failed to bind MAb H3TS. This indicated that the epitope of the B31OspA which binds H3TS is comprised of amino acid side-chains on bothsides of Trp216.

[0181] The 613/625 mutant failed to bind MAbs 105 and H5332, while theother mutants retained their ability to bind these MAbs. This isimportant in light of the data using fusion proteins that indicate thatMAb 105 behaves more like MAb H3TS in terms of its serotype specificityand binding to OspA (Wilske, B. et al., Med. Microbiol. Immunol.,181:191 (1992)). The 613/625 protein has, in addition to the differencesat residues Thr204 and Ser206, changes immediately amino-terminal toTrp216 (Ala214-Gly and Ala215-Lys). The abrogation of reactivity of MAbs105 and H5332 to this protein indicated that the epitopes of OspA whichbind these monoclonal antibodies are comprised of residues on theamino-terminal side of Trp216.

[0182] The two proteins carrying the Asn217-Asp and Gly219-Lysreplacements on the carboxy-terminal side of Trp216 (OspAs 640 and613/640) retained binding to MAbs 105 and H5332; however, they failed toreact with MAb 336, a monoclonal which has been mapped with TrpE-OspAfusion proteins and by chemical cleavage to a more carboxy-terminaldomain. This result may explain why MAb 336 failed to recognize theK48-type of OspA (Group 2).

[0183] It is clear that amino acids Ser204 and Thr206 play an importantpart in the agglutinating epitopes in the region of the B31 OspAflanking Trp216. Replacement of these two residues altered the epitopesof OspA that bind MAbs 105, H3TS and H5332. The ability of the 640changes alone to abolish reactivity of MAb 336 indicated that Thr204 andSer206 are not involved in direct interaction with MAb 336.

[0184] The results indicated that the epitopes of OspA which areavailable to MAbs that agglutinate spirochetes are comprised at least inpart by amino acids in the immediate vicinity of Trp216. Since circulardichroism analysis indicated that the structures of B31 and K48 OspAdiffer very little within this domain, it is unlikely that the changesmade by mutation have radically altered the overall structure of theOspA protein (France, L. L. et al., Biochem. Biophys. Acta, 1120:59(1992); and France et al., Biochem. Biophys Acta, submitted (1993)).This hypothesis is supported by the finding that the recombinant, mutantOspAs exhibit the same high solubility and purification properties asthe parent B31 protein (data not shown).

[0185] In summary, amino acid side-chains at Ser204 and Thr206 areimportant for many of the agglutinating epitopes. However, a limited setof conservative changes at these sites were not sufficient to abolishbinding of all of the agglutinating MAbs. These results suggested thatthe agglutinating epitopes of OspA are distinct, yet may have someoverlap. The results also supported the hypothesis that thesurface-exposed epitope around Trp216 which is thought to be importantfor immune recognition and neutralization is aconformationally-determined and complex domain of OspA.

Example 3 Borrelia Strains and Proteins

[0186] A. Genes Encoding Borrelia Proteins

[0187] The altered OspA polypeptides of the current invention can bepart of a cocktail with other proteins or can be joined to otherproteins to form a chimeric protein. The other polypeptides of thecocktail or chimeric can be derived from any Borrelia. Representativeproteins include OspA, OspB, OspC, OspD, p12, p39, p41 (fla), p66, andp93. Nucleic acid sequences encoding several Borrelia proteins areavailable (see Table II for examples); alternatively, nucleic acidsequences encoding Borrelia proteins can be isolated and characterizedusing methods such as those described below. TABLE II References forNucleic Acid Sequences for Several Proteins of Various Borrelia StrainsStrain OspA K48 X62624 (SID 8)  PGau X62387 (SID 10) DK29 X63412 (SID21) Pko X65599 (SID 25) PTrob X65598 (SID 45) Ip3 X70365 (SID 24) Ip90Kryuchechnikov, V. N. et al., J. Microbiol. Epid. Immunobiol. 12:41-44(1988) (SID 22) 25015 Fikrig, E. S. et al., J. Immunol. 7:2256-2260(1992) (SID 12) B31 Bergstrom, S. etal., Mol. Microbiol. 3:479-486(1989) (SID 6) PKa1 X69606 (SID 42) ZS7 Jonsson, M. et al., Infect.Immun. 60:1845-1853 (1992) (SID 44) N40 Kryuchechnikov, V. N. et al.(SID 43) PHei X65600 (SID 46) ACAI Kryuchechnikov, V. N. et al. (SID 26)PBo X65605 (SID 23)

[0188] B. Isolation of Borrelia Genes

[0189] Nucleic acid sequences encoding full length, lipidated proteinsfrom known Borrelia strains were isolated using the polymerase chainreaction (PCR) as described below. In addition, nucleic acid sequenceswere generated which encoded truncated proteins (proteins in which thelipidation signal has been removed, such as by eliminating the nucleicacid sequence encoding the first 18 amino acids, resulting innon-lipidated proteins). Other proteins were generated which encodedpolypeptides of a particular gene (i.e., encoding a segment of theprotein which has a different number of amino acids than the proteindoes in nature). Using similar methods as those described below, primerscan be generated from known nucleic acid sequences encoding Borreliaproteins and used to isolate other genes encoding Borrelia proteins.Primers can be designed to amplify all of a gene, as well as to amplifya nucleic acid sequence encoding truncated protein sequences, such asdescribed below for OspC, or nucleic acid sequences encoding apolypeptide derived from a Borrelia protein. Primers can also bedesigned to incorporate unique restriction enzyme cleavage sites intothe amplified nucleic acid sequences. Sequence analysis of the amplifiednucleic acid sequences can then be performed using standard techniques.

[0190] Cloning and Sequencing of OspA Genes and Relevant Nucleic AcidSequences

[0191] Borrelia OspA sequences were isolated in the following manner:100 μl reaction mixtures containing 50 mM KCl, 10 mM TRIS-HCl (pH 8,3),1.5 mM MgCl₂, 200 μM each NTP, 2.5 units of TaqI DNA polymerase(Amplitaq, Perkin-Elmer/Cetus) and 100 pmol each of the 5′ and 3′primers (described below) were used. Amplification was performed in aPerkin-Elmer/Cetus thermal cycler as described (Schubach, W. H. et al.,Infect. Immun., 59:1811-1915 (1991)). The amplicon was visualized on anagarose gel by ethidium bromide staining. Twenty nanograms of thechloroform-extracted PCR product were cloned directly into the PC-TAvector (Invitrogen) by following the manufacturer's instructions.Recombinant colonies containing the amplified fragment were selected,the plasmids were prepared, and the nucleic acid sequence of each OspAwas determined by the dideoxy chain-termination technique using theSequenase kit (United States Biochemical). Directed sequencing wasperformed with M13 primers followed by OspA-specific primers derivedfrom sequences, previously obtained with M13 primers.

[0192] Because the 5′ and 3′ ends of the OspA gene are highly conserved(Fikrig, E. S. et al., J. Immunol., 7:2256-2260 (1992); Bergstrom, S. etal., Mol. Microbiol. 3:479-486 (1989); Zumstein, G. et al., Med.Microbiol. Immunol., 181:57-70 (1992)), the 5′ and 3′ primers forcloning can be based upon any known OspA sequences. For example, thefollowing primers based upon the OspA nucleic acid sequence from strainB31 were used:

[0193] 5′-GGAGAATATATTATGAAA-3′ (−12 to+6) (SEQ ID NO: 4); and

[0194] 5′-CTCCTTATTTTAAAGCG-3′ (+826 to+809) (SEQ ID NO: 5). (Schubach,W. H. et al., Infect. Immun, 59:1811-1915 (1991)).

[0195] OspA genes isolated in this manner include those for strains B31,K48, PGau, and 25015; the nucleic acid sequences are depicted in thesequence listing as SEQ ID NO: 6 (OspA-B31), SEQ ID NO: 8 (OspA-K48),SEQ ID NO: 10 (OspA-PGau), and SEQ ID NO: 12 (OspA-25015). An alignmentof these and other OspA nucleic acid sequences is shown in FIG. 17. Theamino acid sequences of the proteins encoded by these nucleic acidsequences are represented as SEQ ID NO: 7 (OspA-B31), SEQ ID NO: 9(OspA-K48), SEQ ID NO: 11 (OspA-PGau), and SEQ ID NO: 13 (OspA-25015).

[0196] The following primers were used to generate specific nucleic acidsequences of the OspA gene: 5′-GTCTGCAAAAACCATGACAAG-3′ (plus strandprimer #369); (SEQ ID NO:14) 5′-GTCATCAACAGAAGAAAAATTC-3′ (plus strandprimer #357); (SEQ ID NO:15) 5′-CCGGATCCATATGAAAAAATATTTATTGGG-3′ (plusstrand primer #607); (SEQ ID NO:16)5′-CCGGGATCCATATGGCTAAGCAAAATGTTAGC-3′ (plus strand primer #584); (SEQID NO:17) 5′-GCGTTCAAGTACTCCAGA-3′ (minus strand primer #200); (SEQ IDNO:18) 5′-GATATCTAGATCTTATTTTAAAGCGTT-3′ (minus strand primer #586); and(SEQ ID NO:19) 5′-GGATCCGGTGACCTTTTAAAGCGTTTTTAAT-3′ (minus strandprimer #1169). (SEQ ID NO:20)

[0197] C. Expression of Proteins From Borrelia Genes

[0198] The nucleic acid sequences described above can be incorporatedinto expression plasmids, using standard techniques, and transfectedinto compatible host cells in order to express the proteins encoded bythe nucleic acid sequences. As an example, the expression of the p12gene and the isolation of p12 protein is set forth.

[0199] Amplification of the p12 nucleic acid sequence was conducted withprimers that included a NdeI restriction site into the nucleic acidsequence. The PCR product was extracted with phenol/chloroform andprecipitated with ethanol. The precipitated product was digested andligated into an expression plasmid as follows: 15 μl (approximately 1μg) of PCR DNA was combined with 2 μl 10×restriction buffer for NdeI(Gibco/BRL), 1 μl NdeI (Gibco/BRL), and 2 μl distilled water, andincubated overnight at 37° C. This mixture was subsequently combinedwith 3 μl 10×buffer (buffer 3, New England BioLabs), 1 μl BamHI (NEB),and 6 μl distilled water, and incubated at 37° for two hours. Theresultant material was purified by preparative gel electrophoresis usinglow melting point agarose, and the band was visualized under long waveultraviolet light and excised from the gel. The gel slice was treatedwith Gelase using conditions recommended by the manufacturer (EpicentreTechnologies). The resulting DNA pellet was resuspended in 25-50 μl of10 mM TRIS-CL (pH 8.0) and 1 mM EDTA (TE). An aliquot of this materialwas ligated into the pET9c expression vector (Dunn, J. J. et al.,Protein Expression and Purification, 1:159 (1990)).

[0200] To ligate the material into the pET9c expression vector, 20-50 ngof p12 nucleic acid sequences cut and purified as described above wascombined with 5 μl 10 One-Phor-All (OPA) buffer (Pharmacia), 30-60 ngpET9c cut with NdeI and BamHI, 2.5 μl 20 mM ATP, 2 μl T4 DNA ligase(Pharmacia) diluted 1:5 in 1×OPA buffer, and sufficient distilled waterto bring the final volume to 50 μl. The mixture was incubated at 12° C.overnight.

[0201] The resultant ligations were transformed into competent DH5-alphacells and plated on nutrient agar plates containing 50 μg/ml kanamycinand incubated overnight at 37° C. DH5-alpha is used as a “storagestrain” for T7 expression clones, because it is RecA deficient, so thatrecombination and concatenation are not problematic, and because itlacks the T7 RNA polyierase gene necessary to express the cloned gene.The use of this strain allows for cloning of potentially toxic geneproducts while minimizing the chance of deletion and/or rearrangement ofthe desired genes. Other cell lines having similar properties may alsobe used.

[0202] Kanamycin resistant colonies were single-colony purified onnutrient agar plates supplemented with kanamycin at 50 μg/ml. A colonyfrom each isolate was inoculated into 3-5 ml of liquid medium containing50 μg/ml kanamycin, and incubated at 37° C. without agitation. PlasmidDNA was obtained from 1 ml of each isolate using a hot alkaline lysisprocedure (Maniatis, T. et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982)).

[0203] Plasmid DNA was digested with EcoRI and BglII in the followingmanner: 15 μl plasmid DNA was combined with 2 μl 10×buffer 3 (NEB), 1 μlEcoRI (NEB), 1 μl BglII (NEB) and 1 μl distilled water, and incubatedfor two hours at 37° C. The entire reaction mixture was subjected toelectrophoresis on an analytical agarose gel. Plasmids carrying the p12insert were identified by the presence of a band corresponding to 925base-pairs (full length p12) or 875 base-pairs (nonlipidated p12). Oneor two plasmid DNAs from the full length and nonlipidated p12 clones inpET9c were used to transform BL21 DE3 pLysS to kanamycin resistance asdescribed by Studier et al., (Methods in Enzymology, Goeddel, D. (Ed.),Academic Press, 185: 60-89 (1990)). One or two transformants of the fulllength and nonlipidated clones were single-colony purified on nutrientplates containing 25 μg/ml chloramphenicol (to maintain pLysS) and 50μg/ml kanamycin at 37° C. One colony of each isolate was inoculated intoliquid medium supplemented with chloramphenicol and kanamycin andincubated overnight at 37° C. The overnight culture was subcultured thefollowing morning into 500 ml of liquid broth with chloramphenicol (25μg/ml) and kanamycin (50 μg/ml) and grown with aeration at 37° C. in anorbital air-shaker until the absorbance at 600 nm reached 0.4-0.7.Isopropyl-thio-galactoside (IPTG) was added to a final concentration of0.5 mM, for induction, and the culture was incubated for 3-4 hours at37° C. as before. The induced cells were pelleted by centrifugation andresuspended in 25 ml of 20 mM NaPO₄ (pH 7.7). A small aliquot wasremoved for analysis by gel electrophoresis. Expressing clones producedproteins which migrated at the 12 kd position.

[0204] A crude cell lysate was prepared from the culture as describedfor recombinant OspA by Dunn, J. J. et al., (Protein Expression andPurification, 1:159 (1990)). The crude lysate was first passed over aQ-sepharose column (Pharmacia) which had been pre-equilibrated in BufferA: 10 mM NaPO₄ (pH 7.7), 10 mM NaCl, 0.5 mM PMSF. The column was washedwith 10 mM NaPO₄, 50 mM NaCl and 0.5 mM PMSF and then p12 was eluted in10 mM NaPO₄, 0.5 mM PMSF with a NaCl gradient from 50-400 mM. p 12eluted approximately halfway through the gradient between 100 and 200 mMNaCl. The peak fractions were pooled and dialyzed against 10 mM NaPO4(pH 7.7), 10 mM NaCl, 0.5 mM PMSF. The protein was then concentrated andapplied to a Sephadex G50 gel filtration column of approximately 50 mlbed volume (Pharmacia), in 10 mM NaPO₄, 200 mM NaCl, 0.5 mM PMSF. p12would typically elute shortly after the excluded volume marker. Peakfractions were determined by running small aliquots of all fractions ona gel. The p12 peak was pooled and stored in small aliquots at −20° C.

Example 4 Generation of Chimeric Nucleic Acid Sequences and ChimericProteins

[0205] A. General Protocol for Creation of Chimeric Nucleic AcidSequences

[0206] The megaprimer method of site directed mutagenesis and amodification were used to generate chimeric nucleic acid sequences(Sarkar and Sommer, Biotechniques, 8(4): 404-407 (1990); Aiyar, A. andJ. Leis, Biotechniques, 14(3): 366-369 (1993)). A 5′ primer for thefirst genomic template and a 3′ fusion oligo are used to amplify thedesired region. The fusion primer consists of a 3′ end of the firsttemplate (DNA that encodes the amino-proximal polypeptide of the fusionprotein), coupled to a 5′ end of the second template (DNA that encodesthe carboxy-proximal polypeptide of the fusion protein).

[0207] The PCR amplifications are performed using Taq DNA polymerase,10×PCR buffer, and MgCl₂ (Promega Corp., Madison, Wis.), and UltrapuredNTPs (Pharmacia, Piscataway, N.J.). One μg of genomic template 1, 5 μlof 10 μM 5′ oligo and 5 μl of 10 μM fusion oligo are combined with thefollowing reagents at indicated final concentrations: 10×Buffer-Mg FREE(1×), MgCl₂ (2 mM), dNTP mix (200 μM each dNTP), Taq DNA polymerase (2.5units), water to bring final volume to 100 μl. A Thermal Cycler (PerkinElmer Cetus, Norwalk, Conn.) is used to amplify under the followingconditions: 35 cycles at 95° C. for one minute, 55° C. for two minutes,and 72° for three minutes. This procedure results in a “megaprimer”.

[0208] The resulting megaprimer is run on a 1×TAE, 4% low-melt agarosegel. The megaprimer band is cut from the gel and purified using thePromega Magic PCR Preps DNA purification system. Purified megaprimer isthen used in a second PCR step. One μg of genomic template 2,approximately 0.5 μg of the megaprimer, and 5 μl of 10 μM 3′ oligo areadded to a cocktail of 10×buffer, MgCl₂, dNTPs and Taq at the same finalconcentrations as noted above, and brought to 100 μl with water. PCRconditions are the same as above. The fusion product resulting from thisamplification is also purified using the Promega Magic PCR Preps DNApurification system.

[0209] The fusion product is then ligated into TA vector and transformedinto E. coli using the Invitrogen (San Diego, Calif.) TA Cloning Kit.Approximately 50 ng of PCR fusion product is ligated to 50 ng of pCRIIvector with 1×Ligation Buffer, 4 units of T4 ligase, and brought to 10μl with water. This ligated product mixture is incubated at 12° C.overnight (approximately 14 hours). Two μl of the ligation productmixture is added to 50 μl competent INC F′ cells and 2 μl betamercaptoethanol. The cells are then incubated for 30 minutes, followedby heat shock treatment at 42° C. for 60 seconds, and an ice quenchingfor two minutes. 450 μl of warmed SOC media is then added to the cells,resulting in a transformed cell culture which is incubated at 37° C. forone hour with slight shaking. 50 μl of the transformed cell culture isplated on LB+50 μg/μl ampicillin plates and incubated overnight at 37°C. Single white colonies are picked and added to individual overnightcultures containing 3 ml LB with ampicillin (50 μg/μl).

[0210] The individual overnight cultures are prepared using Promega'sMagic Miniprep DNA purification system. A small amount of the resultingDNA is cut using a restriction digest as a check. DNA sequencing is thenperformed to check the sequence of the fusion nucleic acid sequence,using the United States Biochemical (Cleveland, Ohio) Sequenase Version2.0 DNA sequencing kit. Three to five μg of plasmid DNA is used perreaction. 2 μl 2M NaOH/2mM EDTA are added to the DNA, and the volume isbrought to 20 μl with water. The mixture is then incubated at roomtemperature for five minutes. 7 μl water, 3 μl 3M NaAc, 75 μl ethanolare added. The resultant mixture is mixed by vortex and incubated forten minutes at −70° C., and then subjected to microcentrifugation. Aftermicrocentrifugating for ten minutes, the supernatant is aspirated off,and the pellet is dried in the speed vac for 30 second. 6 μl water, 2 μlannealing buffer, and 2 μl of 10 μM of the appropriate oligo is thenadded. This mixture is incubated for 10 minutes at 37° C. and thenallowed to stand at room temperature for 10 minutes. Subsequently, 5.5μl of label cocktail (described above) is added to each sample of themixture, which are incubated at room temperature for an additional fiveminutes. 3.5 μl labeled DNA is then added to each sample which is thenincubated for five minutes at 37° C. 4 μl stop solution is added to eachwell. The DNA is denatured at 95° for two minutes, and then placed onice.

[0211] Clones with the desired fusion nucleic acid sequences are thenrecloned in frame in the pET expression system in the lipidated (fulllength) and non-lipidated (truncated, i.e., without first 17 aminoacids) forms. The product is amplified using restriction sites containedin the PCR primers. The vector and product are cut with the same enzymesand ligated together with T4 ligase. The resultant plasmid istransformed into competent E. coli using standard transformationtechniques. Colonies are screened as described earlier and positiveclones are transformed into expression cells, such as E. coli BL21, forprotein expression with IPTG for induction. The expressed protein in itsbacterial culture lysate form and/or purified form is then injected inmice for antibody production. The mice are bled, and the sera collectedfor agglutination, in vitro growth inhibition, and complement—dependentand —independent lysis tests.

[0212] A specific example of chimeric OspA is as follows. Other OspAchimeras can be made using the same method with suitable primers.

[0213] OspA-K48/OspA-PGau

[0214] A chimer of OspA from strain K48 (OspA-K48) and OspA from strainPGau (OspA-PGau) was generated using the method described above. Thischimeric nucleic acid sequence included bp 1-654 from OspA-K48, followedby bp 655-820 from OspA-PGau. Primers used included: the amino-terminalsequence of OspA primer #607 (SEQ ID NO: 16); the fusion primer,5′-AAAGTAGAAGTTTTTGAATCCCATTTTCCAGTTTTTTT-3′ (minus strand primer#668-654) (SEQ ID NO: 27); the carboxy-terminal sequence of OspA primer#586 (SEQ ID NO: 19); and the sequence primers #369 (SEQ ID NO: 14) and#357 (SEQ ID NO: 15). The chimeric nucleic acid sequence is presented asSEQ ID NO: 28; the chimeric protein encoded by this chimeric nucleicacid sequence is presented as SEQ ID NO: 29.

[0215] C. Purification of Proteins Generated by Chimeric Nucleic AcidSequences

[0216] The chimeric nucleic acid sequences described above, as well aschimeric nucleic acid sequences produced by the methods described above,are used to produce chimeric proteins encoded by the nucleic acidsequences. Standard methods, such as those described above in Example 3,concerning the expression of proteins from Borrelia genes, can be usedto express the proteins in a compatible host organism. The chimericproteins can then be isolated and purified using standard techniques.

[0217] Nucleic acid encoding altered versions of OspA can be used togenerate OspA chimeras. In addition, nucleic acid encoding OspA chimerascan be used to generate the altered OspA polypeptides of the presentinvention.

[0218] If the chimeric protein is soluble, it can be purified on aSepharose column. Insoluble proteins can be solubilized in guanidine andpurified on a Ni²⁺ column; alternatively, they can be solubilized in 10mM NaPO₄ with 0.1-1% TRIXON X 114, and subsequently purified over an Scolumn (Pharmacia). Lipidated proteins were generally purified by thelatter method. Solubility was determined by separating both soluble andinsoluble fractions of cell lysate on a 12% PAGE gel, and checking forthe localization of the protein by Coomassie staining, or by Westernblotting with monoclonal antibodies directed to an antigenic polypeptideof the chimeric protein.

Example 5 Generation of OspC/OspA Chimeric Nucleic Acids and ChimericProteins

[0219] A. General Protocol for Creation of Chimeric Nucleic AcidSequences

[0220] A large number of chimeric nucleic acid sequences encodingproteins comprising at least a first and a second polypeptide fromBorrelia burgdorferi were generated. These chimeric nucleic acidsequences were produced such that the encoded chimeric protein compriseda Borrelia burgdorferi OspC polypeptide upstream of (or N-terminal to) aBorrelia burgdorferi OspA polypeptide. The chimeric nucleic acidsequences were also produced such that the nucleic acid encoding onepolypeptide was in the same reading frame as the nucleic acid sequenceencoding the next polypeptide in the chimeric protein.

[0221] The general cloning strategy used to construct the chimericnucleic acid sequences was as follows. The desired fragment of OspC wasamplified using a 5′ primer containing a restriction site suitable forcloning the resultant product into a vector of interest and a 3′ primercontaining a restriction site suitable for ligating the OspC fragment tothe OspA fragment. The OspC product was cloned into a suitable vector.For-the OspA portion of the chimeric nucleic acid, the desired OspAfragment was amplified using a 5′ primer containing a restriction sitefor ligating the resultant OspA fragment to the OspC fragment and a 3′primer containing a restriction site suitable for cloning the resultantOspA product into the vector with the OspC product. The use of arestriction site to allow ligation of the OspC and OspA fragmentsresults in the insertion of 0 to about 3 amino acids between the OspCand OspA fragments.

[0222] A specific example of such a construction follows. It isunderstood that other suitable restriction sites could be used with noor only routine experimentation. The resultant OspC/OspA Chimer couldhave, therefore, the addition of 0 to about 3 amino acids or morebetween the OspC and OspA fragments, depending on the restriction siteused.

[0223] For the OspC portions of the chimeric nucleic acids, desiredfragments of OspC genes from various strains or genospecies were PCRamplified using a 5′ primer containing an NdeI site and a 3′ primercontaining a NcoI site and a BamHI site. The amplified OspC product wasthen cloned into the NdeI and BamHI sites of the T7 promoter drivenexpression vector, pET9c. For the OspA portion of the chimeric nucleicacid, desired fragments of OspA genes from a strain of interest orgenospecies of interest were PCR amplified using a 5′ primer containingan NcoI site and a 3′ primer containing a BamHI site. This OspA portioncould then be directly cloned into the NcoI and BamHI sites of the pET9cvector containing the desired OspC sequence, thereby producing thedesired OspC-OspA construct. By including the sequence for the NcoIrestriction site in the primers, a nine nucleotide linker sequenceencoding the amino acids Ser-Met-Ala was produced at the junctionbetween the N-terminal OspC sequence and the C-terminal OspA sequence.The use of the NcoI restriction enzyme (CCATGG) in this cloning strategywas a suitable choice as Borrelia is an AT-rich organism which possessesonly a few NcoI sites in its genome. One of ordinary skill in the artwould know that different restriction sites wold be utilized and wouldknow how to generate the chimeric OspC/A constructs for use insubsequent alterations described herein with no or only routineexperimentation.

[0224] As an example, OspC-OspA chimeric nucleic acids which containnonlipidated OspC B31 were generated using the following primers:(5′OspC-NdeI): 5′-GT CAT ATG GCT TGT AAT AAT TCA GGG AAA GA-3′; and (SEQID NO:91) (3′OspC-NcoI): 5′-T TTC CAT GGA AGG TTT TTT TGG ACT TTC TG-3′.(SEQ ID NO:92)

[0225] For OspC-OspA chimeric nucleic acids which contain nonlipidatedOspA B31, the following primers were used: (5′OspA-NcoI:) 5′-TT TCC ATGGCC AAG CAA AAT GTT AGC AGC C-3′; and (SEQ ID NO:93) (3′OspA-BamHI):5′-TAA GGA TCC TTA TTT TAA AGC GTT TTT-3′. (SEQ ID NO:94)

[0226] Lipidated versions of the OspC/OspA chimeras can be constructedby designing primers to amplify the a portion of the template thatincludes the lipidation signal sequence or by generating a nucleic acidconstruct with a suitable lipidation signal sequence. The leadersequence comprising a lipidation signal can be, for example, from a geneencoding the OspA, OspB or OspC polypeptides.

[0227] B. Protein Expression

[0228] As described in the previous examples, it is possible to expressand purify Borrelia proteins or polypeptides, for example, OspApolypeptides, OspC polypeptides, chimeric OspC/OspA polypeptides andpolypeptides comprising the OspA alterations described herein. This isaccomplished by incorporating the desired nucleic acid sequence, whichencodes the protein of choice, into an expression plasmid, for example,using standard techniques. This expression plasmid can then betransfected into a compatible host cell in order to express the desiredprotein.

[0229] For example, the purified OspA, OspC or OspC/OspA chimericproteins that were used to immunize mice and in the ELISA testsdescribed below, were generated and purified by cloning either OspA,OspC or OspC/OspA chimeric nucleic acid sequences, in frame, into thepET expression plasmid. The expression plasmid was then transfected intothe compatible expression cell line Escherichia coli strain BL21 (pLysS)or strain B834 (DE3). The BL21 or B834 cells were grown in 10 mL LBmedia (5 g/L NaCl, 10 g/L tryptone, 5 g/L yeast extract, 25 mg/Lchloramphenicol and 50 mg/L ampicillin) at 37° C., with shaking. Whenthe optical density at 600λ reached 0.3-0.4 units, recombinant proteinexpression was induced by adding IPTG (isopropylB-D-thiogalactopyranoside) to a final concentration of 0.5 mM and thecells were grown for an additional three hours. The cultures wereharvested by centrifugation at 3800×g for five minutes. The cells wereresuspended in 20 mM NaPO₄, pH 7.7, and stored at −20° C. overnight.Once thawed, the crude extracts were incubated with DNase (2 μg/mL) inthe presence of 2.5 mM MgCl₂ at room temperature for thirty minutes andthen spun at 14,000 rpm (Eppendorf 5417C) for five minutes.

[0230] To purify the OspC proteins described below, the crude extractsfrom the OspC-expressing cells were loaded onto an anion exchange column(Q Sepharose Fast Flow, 2.2×10 cm, Pharmacia) which had beenpre-equilibrated with 20 mM Tris-Cl, pH 9.3. The column was washed inthe same buffer (20 mM Tris-Cl, pH 9.3) which eluted the OspC protein.The wash fractions that contained OspC were concentrated using Amicon10K and then were dialyzed with a solution containing 20 mM NaPO₄, pH8.0, and 250 mM NaCl. The partially purified OspC was then passed over aNi²⁺ metal affinity column (Chelating Sepharose Fast Flow 2.2×10 cm,Pharmacia) equilibrated with 20 mM NaPO₄, pH 8.0, and 250 mM NaCl. Thecolumn was washed using a decreasing pH gradient of 20 mM sodium aceticacid and 250 mM NaCl and the bound OspC eluted around pH 5.7. The OspCfractions were then concentrated by ultrafiltration and stored at −70°C.

[0231] For purification of OspA proteins, the same procedure wasfollowed, except that the dialysis step, after the Amicon 10K cutoff,was done in 20 mM NaPO₄, pH 6.0. The partially purified OspA was thenapplied to a cation exchange column (S Sepharose Fast Flow 2.2×10 cm,Pharmacia) equilibrated with 20 nM NaPO₄, pH 6.0. The column was washedusing an increasing NaCl gradient from 0 to 100 mM. The OspA-containingfractions were concentrated by ultrafiltration and stored at −70° C.

[0232] As previously indicated, both lipidated and non-lipidated (e.g.,truncated, lacking the first 17 amino acids) forms of OspC, OspA andOspC/OspA chimeric proteins were generated.

[0233] The above-described techniques were also used to express proteinscomprising the altered OspA polypeptides of the present invention.

[0234] C. Immunization of Mice and Serologic Characterization UsingELISA (Enzyme-Linked Immunosorbent Assay)

[0235] Immunization of Mice

[0236] Mice, either C3H-J or ICR, were immunized with 3 μg of lipidatedOspC/OspA chimeric protein or 6 μg of non-lipidated OspC/OspA chimer in100 mL of aluminum hydroxide adjuvant (concentration of 1.8 mg/mL) by(SC) subcutaneous injection. As a negative control, mice were immunizedwith 100 mL of aluminum hydroxide adjuvant only. All mice received atotal of three injections which were given at two week intervals. Oneweek after the final immunization, blood was drawn from each mouse(including negative control mice) and the serum was tested for IgGreactivity using the ELISA method described below, for the presence ofanti-OspA antibodies to three different purified OspA proteins (Borreliaburgdorferi sensu stricto (B31), Borrelia garinii (K48) and Borreliaafzelii (PGau). The sera was tested at a dilution of 1:1000.

[0237] Mice were immunized with the chimeric proteins described in TableIII. TABLE III Chimeric Proteins Used to Immunize Mice SEQ SEQ ID NO: IDNO: Description (nucleic (poly- Name (amino acid) acid) peptide) FIG.No: OspA OspA-B31(18-273)  6  7 22, 23 OspC OspC-B31(19-211) * * 22, 23OspC2- OspC-C2(19-204)/ 59 60 22, 23 OspA OspA-B31(18-273) ¹lipOspAP/OspA-PGau(1-217)/ 49 50 24, 25 Bo OspA-Bo(218-273) ¹lipOspAB/POspA-B31(1-216)/ * * 24, 25 OspA-Pko(217-273) OspC- OspC-B31(19-211)/ 6566 24, 25, OspAB/P OspA-B31(18-216)/ 27, 28, 29 OspA-Pko(217-273)OspCB31- OspC-B31(19-211)/ 55 56 26, 27, OspAB31 OspA-B31(18-273) 28, 29OspC2- OspC-C2(19-204)/ 59 60 26, 27 OspAB31 OspA-B31(18-273) ¹lip OspAOspA-K48(1-217)/ * * 27 K/T OspA-Tro(218-273) ¹lip OspC-OspC-B31(1-211) * * 26 B31 OspCB31- OspC-B31(19-211)/ 87 88 28, 29OspABPBP OspA-B31(30-150)/ OspA-Pko(151-179)/ OspA-B31(180-216) (190 Ndeletion)/ OspA-Pko(217-273) B31/B31/Pko

[0238] Immobilization of Antigen Onto ELISA Plates

[0239] A solution of purified recombinant OspC or OspA protein from eachof the Borrelia burgdorferi strains B31 (Borrelia burgdorferi sensustricto), K48 (Borrelia garinii) and PGau (Borrelia afzelii) was addedto sodium phosphate buffer, pH 9.0, and was used to coat a commercialmicrowell plate (MaxiSorp®, Nunc). The coating procedure was as follows:100 μl of a solution containing the appropriate OspA or OspC protein(made up at a concentration of 250 ng/ml in the following coatingbuffer: 100 mM Bis-Tris propane, pH 9.7) was added to each well of amicrotiter plate which was incubated for one hour at 37° C. The antigensolution was removed from the wells, the plate was washed three timeswith phosphate buffered saline (PBS) pH 9.0, and 300 μl of blockingbuffer solution was added (3% dry milk, 0.1% polyoxyethylenesorbitan(referred to herein as Tween 20™), 0.02% NaN₃ in 100 nM Bis-Trispropane, pH 9.7). Following a one hour incubation at 37° C., the plateswere washed four times with TBS-Tween 20™ wash buffer (20 mM Tris-Cl, pH7.5, 136 mM NaCl, 0.1% Tween 20™ and 0.02% NaN₃) and then were allowedto dry. The plates were then wrapped in plastic and stored at 4° C.until they were used.

[0240] ELISA (Enzyme-Linked Immunosorbent Assay) Tests

[0241] The standard procedure for the ELISA tests was as follows: mouseserum was diluted 1:1000 in sample dilution buffer (1% dry milk, 136 mMNaCl, 0.1% Tween 20™, 0.02% NaN₃ in 20 mM Tris-Cl, pH7.5) and 100 μl ofthe diluted serum was added to the ELISA microtiter plate wells that hadbeen coated with antigen as described above. Following incubation for 1hour at 37° C., the samples were removed and the plates were washed fourtimes in TBS-Tween™ (20 mM Tris-Cl, pH 7.5; 136 mM NaCl; 0.1% Tween 20™and 0.02% NaN₃). For the secondary antibody, goat anti-mouse antiseraconjugated to alkaline phosphatase-specific for either IgM (Fc) or IgG(Fab), (Jackson Immuno Research Laboratories) was diluted 1:750 insample dilution buffer (1% dry milk, 136 mM NaCl, 0.1% Tween 20™, 0.02%NaN₃ in 20 mM Tris-Cl, pH7.5) and 100 μl of the diluted secondaryantibody was added to each well. Following incubation for thirty minutesat 37° C., the plates were washed three times with TBS-Tween™ and 100 μlof Phosphatase Substrate solution (5 mg of p-nitrophenylphosphatetablets dissolved in 1×diethanolamine substrate buffer to yield a 2mg/ml solution—Kirkegaard Perry Laboratory) was added to each well. Theplates were incubated for thirty minutes at 37° C. and 100 μl of stopsolution (5% EDTA) was added to each well. The absorbance at 405 nm wasread on a microplate reader (Dynatech). A sample was considered positiveif it produced an average absorbance greater than the mean of thenegative controls plus three standard deviations.

[0242] Previous work has demonstrated that it is the carboxy-terminalregion of OspA that contains the antigenic sites that provide theimmunoprotective response. Thus, in addition to the ELISA test describedabove, a modified ELISA was performed (herein referred to as theProtective ELISA Test), wherein the purified N-terminal region of B31OspA (amino acids 18-139) was used in excess to block any antibodiespresent in the mouse serum that had specificity to this N-terminal OspAregion. These protective ELISA tests were performed as above, exceptthat 80 μg/ml of a purified B31 OspA fragment (amino acids 18-139) wasadded to the diluted mouse serum prior to adding the sera to theantigen-coated ELISA microtiter plate wells.

[0243] Results of ELISA Tests

[0244] Using the above-described ELISA tests, it was demonstrated thatmice immunized with a non-lipidated OspC/OspA chimeric protein(OspC2-OspA—composed of OspC (a.a. 19-204 from strain C2)/OspA (a.a.18-273 from strain B31) (SEQ ID NO: 60) produced an immune response bothto OspA and OspC that was comparable to the immune response generated tonon-lipidated OspA (OspA—a.a. 18-273 from strain B31) and non-lipidatedOspC (OspC—a.a. 19-211 from strain B31) control proteins (FIG. 22). Asindicated in FIG. 22 and described above, mice were immunized with OspA,OspC or OspC2-OspA proteins and immune responses of the sera weremeasured against B31 OspA antigen (stippled bars) and B31 OspC antigen(solid bars).

[0245] Using the above-described Protective ELISA Test, it was alsoshown that mice immunized with the same non-lipidated OspC/OspA chimericprotein (OspC2-OspA—composed of OspC (a.a. 19-204 from strain C2)/OspA(a.a. 18-273 from strain B31) (SEQ ID NO: 60) produced an immuneresponse to the C-terminal portion of OspA that was comparable to theimmune response generated to the C-terminal portion of a non-lipidatedOspA (OspA—a.a. 18-273 from strain B31) control protein (FIG. 23). Asindicated in FIG. 23, mice were immunized with OspA, OspC or OspC2-OspAproteins and immune responses of the sera were measured against B31 OspAantigen. The protective antibody response to B31 OspA antigen isindicated in the stippled bars.

[0246] Thus, these results clearly demonstrate that non-lipidatedchimeric OspC/OspA proteins are able to induce immune responses in micethat are comparable to the immune response generated againstnon-lipidated OspC and OspA control proteins.

[0247] It had been previously thought that the lipidation signals thatare present on Borrelia burgdorferi outer surface proteins were requiredfor immunogenicity and that OspC and OspA proteins that lacked thislipidation signal would be less or non-immunogenic. To test this idea,mice were immunized with a non-lipidated OspC/OspA chimeric protein(OspC-OspAB/P—composed of OspC (a.a. 19-211 from strain B31)/OspA (a.a.18-216 from strain B31)/OspA (a.a. 217-273 from strain Pko)(SEQ ID NO:66) as well as two lipidated OspA proteins, lipOspAP/Bo (composed ofOspA (a.a. 1-217 from strain PGau)/OspA (a.a. 218-273 from strain Bo))and lipOspAB/P (composed of OspA (a.a. 1-216 from strain B31)/OspA (a.a.217-273 from strain Pko)) and were subjected ELISA tests. Mice immunizedwith the non-lipidated OspC/OspA chimeric protein (OspC-OspAB/P)produced an immune response to OspA from each of the Borreliaburgdorferi strains B31 (Borrelia burgdorferi sensu stricto), K48(Borrelia garinii) and PGau (Borrelia afzelii), that was equivalent orgreater than the immune response generated to the two lipidated OspAcontrol proteins (lipOspAP/Bo and lipOspAb/P) (FIG. 24).

[0248] Similar results to these were obtained using the Protective ELISATest described above. Mice immunized with the non-lipidated OspC/OspAchimeric protein (OspC-OspAB/P) produced an immune response to theC-terminal region of OspA from each of the Borrelia burgdorferi strainsB31 (Borrelia burgdorferi sensu stricto), K48 (Borrelia garinii) andPGau (Borrelia afzelii), that was equivalent or greater than the immuneresponse generated to the C-terminal region of OspA from the twolipidated OspA control proteins (lipOspAP/Bo and lipOspAb/P) (FIG. 25).

[0249] In addition to the comparisons between non-lipidated OspC/OspAchimeric proteins and lipidated OspA control proteins, experiments werealso performed to compare non-lipidated OspC/OspA chimeric proteins witha lipidated OspC control protein (FIG. 26). Mice that were immunizedwith either the non-lipidated OspC/OspA chimeric protein OspCB31-OspAB31(composed of OspC (a.a. 19-211 from strain B31)/OspA (a.a. 18-273 fromstrain B31) (SEQ ID NO: 56) or the non-lipidated OspC/OspA chimericprotein OspC2-OspAB31 (composed of OspC (a.a. 19-204 from strainC2)/OspA (a.a. 18-273 from strain B31) (SEQ TD NO: 60) produced animmune response to OspC derived from the Borrelia burgdorferi strain B31that was comparable to the immune response produced by a lipidated OspCcontrol protein (lip OspC-B31−composed of OspC (a.a. 1-211 from strainB31)) (FIG. 26).

[0250] Thus, these results clearly demonstrate that non-lipidatedchimeric OspC/OspA proteins are able to induce immune responses againstOspA and OspC that are comparable to the immune response generatedagainst OspA and OspC using lipidated OspA or OspC control proteins. Theuse of unlipidated forms of these proteins as vaccine immunogens ordiagnostic antigens is highly desirable because the product yield ismuch greater and the proteins are much easier to purify. For thesereasons, the production of these proteins is less expensive.

[0251] The OspC/OspA chimeric proteins of the present invention are alsoable to generate immune responses against OspA proteins that are derivedfrom strains that are not represented in the chimeric protein. Miceimmunized with the OspC/OspA chimeric proteins, OspCB31-OspAB31 (SEQ IDNO: 56) and OspC2-OspAB31 (SEQ ID NO: 60), are not only able to generateimmune responses that recognize OspA derived from strain B31 (Borreliaburgdorferi sensu stricto), but also recognize OspA derived from strainK48 (Borrelia garinii) and strain PGau (Borrelia afzelii) (FIG. 27). Forcomparison, mice were also immunized with the lipidated OspA chimericprotein, Lip OspA K/T (composed of OspA (a.a. 1-217 from strainK48)/OspA (a.a. 218-273 from strain Tro)) (FIG. 27).

[0252] Additional antibody responses to OspA derived from strain B31(Borrelia burgdorferi sensu stricto), strain K48 (Borrelia garinii) andstrain PGau (Borrelia afzelii) are also presented for sera from miceimmunized with other OspC/OspA chimeric proteins. Thus, FIG. 28 presentsthe ELISA results from mice immunized with either OspCB31-OspAB/P (SEQID NO: 66), OspCB31-OspABPBP (SEQ ID NO: 88) or OspCB31-OspAB31 (SEQ IDNO: 56). In each case, sera from the immunized mice was tested againstOspA derived from each of strain B31 (Borrelia burgdorferi sensustricto), K48 (Borrelia garinii) and PGau (Borrelia afzelii). In allcases, a strong immune response was generated (FIG. 28). As with thepreviously described OspC/OspA chimeric proteins, the three OspC/OspAchimeric proteins used to immunize the mice in FIG. 27 also elicited astrong immune response to the C-terminal region of OspA when examinedusing the Protective ELISA Test described above (FIG. 29).

[0253] The above-described techniques are also used to immunize mice andto serologically characterize the immune response against the proteinscomprising the altered OspA polypeptides of the present invention.

[0254] Tick Challenge of Immunized Mice

[0255] Mice, either C3H-J or JCR, that had been immunized as describedabove, were also challenged with either laboratory-infected nympha orfield nympha. The immunized mice were placed in isolation cages and eachmouse received 5-10 nymphs. All of the nymphs were collected at countedafter 6 days. Four week after challenge, the mice were bled and sera wastested using commercially-available Western blot strips to Borreliaburgdorferi sensu stricto strain B31 (MarDx strips) and/or Borreliagarinii (MRL strips). Eight weeks after challenge, the mice were bled,sera was tested again by Western blot and ear punch and bladder sampleswere cultured. As a positive control, mice which had been immunized withonly aluminum hydroxide adjuvant, as described above, were subjected tothe same challenge.

[0256] The results of the tick challenge studies (Table IV) demonstratethat while immunization with lipidated OspC protein was unable toprotect the mice, as evidenced by a positive Western blot signal (in 4out of 5 mice), immunization with two different OspC/OspA chimericproteins (SEQ ID NO: 56 and SEQ ID NO: 62) did provide protection, asindicated by the absence of Western blot signal (in 0 out of 8 mice and0 out of 3 mice) (Table IV). The sham positive control showed that thechallenge by the ticks was successful in all cases, as evidenced by 100%positive signal in Western blots (Table IV). Results from the tickchallenge experiments are shown in Table IV. TABLE IV Effect ofVaccination on Transmission of Borrelia from Ticks SeroconversionSeroconversion Vaccine (Western Blots) (Western Blots) Candidate MouseTick-nymph Vaccinated Sham OspC1- C3H-J Long Island 0+/8 8+/8 OspAB31OspC2- C3H-J Long Island 0+/3 4+/4 OspAB31 Lip OspC12 ICR Long Island4+/5 5+/5

[0257] The above-described techniques are also used to measure theability of mice immunized with proteins comprising the altered OspApolypeptides of the present invention to resist or respond totransmission of Borrelia from ticks.

Example 6 Generation of OspA M1, M2, M3, J1, J2 and J3 constructs usingsite directed mutagenesis and PCR

[0258] All constructs were made using Stratagene's QuikChangesite-directed mutagenesis kit. Site-directed mutagenesis was performedusing Pfu Turbo DNA polymerase II and a thermal temperature cycler. PfuTurbo DNA polymerase replicates both plasmid strands with high fidelityand without displacing the mutant oligonucleotide primers. The basicprocedure used a supercoiled double-stranded DNA vector (pET 9c for allconstructs) with an insert of interest and two synthetic oligonucleotideprimers containing the desired mutation(s). The oligonucleotide primers,each complementary to opposite strands of the vector, were extendedduring the temperature cycling by the Pfu Turbo DNA polymerase.Incorporation of the oligonucleotide primers created a mutated plasmidwhich contained staggered nicks. Following temperature cycling, thelinear product was treated with the restriction enzyme Dpn I, which isspecific for methylated DNA. DNA isolated from most E. coli strains isdam methylated and therefore is susceptible to Dpn I digestion.Digestion with Dpn I therefore destroyed the original template DNAleaving only the nicked plasmid DNA (which was not methylated)containing the desired mutation(s). This nicked vector DNA (containingthe desired mutation(s)) was then transformed into competent E. coliwhich were plated on antibiotic-containing plates. Colonies containingthe plasmid (which encodes for antibiotic resistance in addition to thealtered OspA polypeptide) were grown and plasmids were purified andsequenced to confirm that they possessed the desired mutation(s).

[0259] 1) M1, M2 and M3 Mutants

[0260] The mutations described herein are made using nucleic acids(e.g., polynucleotides) encoding OspA polypeptides or fragments from anystrain of Lyme Disease-causing Borrelia, such as Borrelia burgdorferisensu stricto, Borrelia afzelii or Borrelia garinii. As an example, thegeneration of the M3 mutations in an OspA chimera, called “BPBP”, isdescribed below. The creation of the construct OspA BPBP M3 was asfollows. BPBP is a chimeric OspA polypeptide, wherein residues 1-164 arefrom OspA of B31, residues 165-179 are from OspA of Pko or PGau,residues 180-216 are from OspA of B31 and residues 217-273 are from OspAof Pko (wherein the numbering is as shown in SEQ ID NO: 7).

[0261] The first step in creating this construct was to make theconstruct BPBP M1. The M1 mutation consists of a mutation of codon 139(aga) of OspA from the amino acid arginine to the amino acid methionine(codon atg). A PCR reaction was set up as described above containing apolynucleotide encoding BPBP as the template DNA and the followingoligonucleotide primers:

[0262] a) 5′ R139M:

[0263] 5′ gaa aaa ata ata aca atg gca gac gga acc 3′ (SEQ ID NO: 117).

[0264] b) 3′ R139M:

[0265] 5′ ggt tcc gtc tgc cat tgt tat tat ttt ttc 3′ (SEQ ID NO: 118)

[0266] The PCR reaction also contained reaction buffer, 10 ng of theBPBP DNA template, 125 ng of each oligonucleotide primer, and the dNTPmix. The parameters for the PCR were as outlined in Table V. TABLE VParameters for PCR Segment cycles temp time 1  1 95° C. 30 sec. 2 18 95°C. 30 sec. 3 50° C.  1 min. 4 68° C. 12 min.

[0267] Following PCR, the product was transformed into competent E. coliXL1-Blue cells. Colonies formed on the selective agar plates (containingkanamycin) were grown and plasmids were purified and sequenced toconfirm that they possessed the desired mutation. The construct BPBP MIwas then used as template to make the plasmid BPBP M2, which in additionto the R139M mutation, also contains a mutation that changes theglutamic acid codon (gag) at position 160 to the codon (tat) whichencodes for tyrosine. The oligonucleotide primers used to generate thisplasmid were:

[0268] a) 5′ E160Y

[0269] 5′ gga aaa gct aaa tat gtt tta aaa ggc 3′ (SEQ ID NO: 119)

[0270] b) 3′ E160Y

[0271] 5′ gcc ttt taa aac ata ttt age ttt tcc 3′ (SEQ ID NO: 120)

[0272] The parameters for the PCR were as outlined in Table VII. Thefinal mutation, M3, which alters a lysine residue at position 189 to amethionine residue was performed using BPBP M2 as a DNA template. Theoligonucleotide primers used to generate this plasmid were:

[0273] a) 5′ K189M

[0274] 5′ gtt act tta age atg aat att tca aaa tc 3′ (SEQ ID NO: 121)

[0275] b) 3′ K189M

[0276] 5′ ga ttt tga aat att cat get taa agt aac 3′ (SEQ ID NO: 122)

[0277] This final mutation yielded the desired construct, BPBP M3.

[0278]2) J1, J2 and J3 Mutants

[0279] J1, J2 and J3 mutants were made using the same protocol describedfor the generation of the M1, M2 and M3 mutants. Such mutants can bemade using nucleic acids (e.g., polynucleotides) encoding OspApolypeptides fragments from any strain of Lyme Disease-causing Borrelia,such as Borrelia burgdorferi sensu stricto, Borrelia afzelii or Borreliagarinii.

[0280] The oligonucleotide primers which were used to generate the J1mutation (which contains a Y165F mutation (codon tat to ttt) and a V166Tmutation (codon gtt to act)) were as follows:

[0281] a) 5′ B31 YV-FT

[0282] 5′ gag gtt tta aaa ggc ttt act ctt gaa gga act c 3′ (SEQ ID NO:123)

[0283] b) 3′ B31 YV-FT

[0284] 5′ gag ttc ctt caa gag taa agc ctt tta aaa cct g 3′ (SEQ ID NO:124)

[0285] The oligonucleotide primers which were used to generate the J2mutation (which contains a T170K mutation (codon act to aag)) were asfollows:

[0286] a) 5′B31 T-K

[0287] 5′ tct tga agg aaa gct aac tgc tg 3′ (SEQ ID NO: 125)

[0288] b) 3′ B31 T-K

[0289] 5′ cag cag tta gct ttc ctt caa ga 3′ (SEQ ID NO: 126)

[0290] To generate the J3 mutant (which contains a Y165F mutation (codontat to ttt), a V166T mutation (codon gtt to act) and a T170K mutation(codon act to aag)), the template containing the J1 mutations was usedwith the oligonucleotide primers for generating the J2 mutation (5′ B31T-K (SEQ ID NO: 125) and 3′ B31 T-K (SEQ ID NO: 126)).

[0291] The altered OspA polypeptides described herein are expressed,used to immunize mice and characterized using ELISA as described in theExperiments above.

[0292] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims.

1 130 1 23 DNA Artificial Sequence Oligonucleotide Primer 1 cttaatgactctgacactag tgc 23 2 30 DNA Artificial Sequence Oligonucleotide Primer 2gctactaaaa aaaccgggaa atggaattca 30 3 33 DNA Artificial SequenceOligonucleotide Primer 3 gcagcttggg attcaaaaac atccacttta aca 33 4 18DNA Artificial Sequence Oligonucleotide Primer 4 ggagaatata ttatgaaa 185 17 DNA Artificial Sequence Oligonucleotide Primer 5 ctccttattt taaagcg17 6 822 DNA Borrelia burgdorferi CDS (1)...(822) 6 atg aaa aaa tat ttattg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu LeuGly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gttagc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val SerSer Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atgaaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met LysVal Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta attgca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile AlaThr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat ggatct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly SerGly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa ttaaca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu ThrIle Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gatggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp GlyLys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tcaaca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser ThrGlu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ataaca aga gca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile ThrArg Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gatgga tct gga aaa gct aaa gag 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp GlySer Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa ggc tat gtt ctt gaagga act cta act gct gaa aaa aca 528 Val Leu Lys Gly Tyr Val Leu Glu GlyThr Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga actgtt act tta agc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr ValThr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaactt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu LeuAsn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tggaat tca ggc act tca act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp AsnSer Gly Thr Ser Thr Leu Thr 210 215 220 att act gta aac agt aaa aaa actaaa gac ctt gtg ttt aca aaa gaa 720 Ile Thr Val Asn Ser Lys Lys Thr LysAsp Leu Val Phe Thr Lys Glu 225 230 235 240 aac aca att aca gta caa caatac gac tca aat ggc acc aaa tta gag 768 Asn Thr Ile Thr Val Gln Gln TyrAsp Ser Asn Gly Thr Lys Leu Glu 245 250 255 ggg tca gca gtt gaa att acaaaa ctt gat gaa att aaa aac gct tta 816 Gly Ser Ala Val Glu Ile Thr LysLeu Asp Glu Ile Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 7 273 PRTBorrelia burgdorferi 7 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile LeuAla Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu LysAsn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val SerLys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val AspLys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly ValLeu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr IleSer Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp GlyLys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser SerThr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys IleIle Thr Arg Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile LysSer Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Gly TyrVal Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val ValLys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys Ser GlyGlu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala ThrLys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 IleThr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235240 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245250 255 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu260 265 270 Lys 8 825 DNA Borrelia burgdorferi CDS (1)...(825) 8 atg aaaaaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys LysTyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aagcaa aat gtt agc agc ctt gat gaa aaa aat agc gtt tca gta 96 Cys Lys GlnAsn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat tta cctggt gga atg aca gtt ctt gta agt aaa gaa aaa gac aaa 144 Asp Leu Pro GlyGly Met Thr Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 gac ggt aaa tacagt cta gag gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr SerLeu Glu Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaaaac aac ggt tct gga aca ctt gaa ggt gaa aaa 240 Gly Thr Ser Asp Lys AsnAsn Gly Ser Gly Thr Leu Glu Gly Glu Lys 65 70 75 80 act gac aaa agt aaagta aaa tta aca att gct gat gac cta agt caa 288 Thr Asp Lys Ser Lys ValLys Leu Thr Ile Ala Asp Asp Leu Ser Gln 85 90 95 act aaa ttt gaa att ttcaaa gaa gat gcc aaa aca tta gta tca aaa 336 Thr Lys Phe Glu Ile Phe LysGlu Asp Ala Lys Thr Leu Val Ser Lys 100 105 110 aaa gta acc ctt aaa gacaag tca tca aca gaa gaa aaa ttc aac gaa 384 Lys Val Thr Leu Lys Asp LysSer Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aag ggt gaa aca tct gaaaaa aca ata gta aga gca aat gga acc aga 432 Lys Gly Glu Thr Ser Glu LysThr Ile Val Arg Ala Asn Gly Thr Arg 130 135 140 ctt gaa tac aca gac ataaaa agc gat gga tcc gga aaa gct aaa gaa 480 Leu Glu Tyr Thr Asp Ile LysSer Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa gac tttact ctt gaa gga act cta gct gct gac ggc aaa 528 Val Leu Lys Asp Phe ThrLeu Glu Gly Thr Leu Ala Ala Asp Gly Lys 165 170 175 aca aca ttg aaa gttaca gaa ggc act gtt gtt tta agc aag aac att 576 Thr Thr Leu Lys Val ThrGlu Gly Thr Val Val Leu Ser Lys Asn Ile 180 185 190 tta aaa tcc gga gaaata aca gtt gca ctt gat gac tct gac act act 624 Leu Lys Ser Gly Glu IleThr Val Ala Leu Asp Asp Ser Asp Thr Thr 195 200 205 cag gct act aaa aaaact gga aaa tgg gat tca aaa act tcc act tta 672 Gln Ala Thr Lys Lys ThrGly Lys Trp Asp Ser Lys Thr Ser Thr Leu 210 215 220 aca att agt gtg aatagc caa aaa acc aaa aac ctt gta ttc aca aaa 720 Thr Ile Ser Val Asn SerGln Lys Thr Lys Asn Leu Val Phe Thr Lys 225 230 235 240 gaa gac aca ataaca gta caa aaa tac gac tca gca ggc acc aat cta 768 Glu Asp Thr Ile ThrVal Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu 245 250 255 gaa ggc aaa gcagtc gaa att aca aca ctt aaa gaa ctt aaa aac gct 816 Glu Gly Lys Ala ValGlu Ile Thr Thr Leu Lys Glu Leu Lys Asn Ala 260 265 270 tta aaa taa 825Leu Lys * 9 274 PRT Borrelia burgdorferi 9 Met Lys Lys Tyr Leu Leu GlyIle Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val SerSer Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Gly MetThr Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 Asp Gly Lys Tyr Ser LeuGlu Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys AsnAsn Gly Ser Gly Thr Leu Glu Gly Glu Lys 65 70 75 80 Thr Asp Lys Ser LysVal Lys Leu Thr Ile Ala Asp Asp Leu Ser Gln 85 90 95 Thr Lys Phe Glu IlePhe Lys Glu Asp Ala Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr LeuLys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly GluThr Ser Glu Lys Thr Ile Val Arg Ala Asn Gly Thr Arg 130 135 140 Leu GluTyr Thr Asp Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160Val Leu Lys Asp Phe Thr Leu Glu Gly Thr Leu Ala Ala Asp Gly Lys 165 170175 Thr Thr Leu Lys Val Thr Glu Gly Thr Val Val Leu Ser Lys Asn Ile 180185 190 Leu Lys Ser Gly Glu Ile Thr Val Ala Leu Asp Asp Ser Asp Thr Thr195 200 205 Gln Ala Thr Lys Lys Thr Gly Lys Trp Asp Ser Lys Thr Ser ThrLeu 210 215 220 Thr Ile Ser Val Asn Ser Gln Lys Thr Lys Asn Leu Val PheThr Lys 225 230 235 240 Glu Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser AlaGly Thr Asn Leu 245 250 255 Glu Gly Lys Ala Val Glu Ile Thr Thr Leu LysGlu Leu Lys Asn Ala 260 265 270 Leu Lys 10 822 DNA Borrelia burgdorferiCDS (1)...(822) 10 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcctta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala LeuIle Ala 1 5 10 15 tgc aag caa aat gtt agc agc ctt gat gaa aaa aac agcgct tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser AlaSer Val 20 25 30 gat ttg cct ggt gag atg aaa gtt ctt gta agt aaa gaa aaagac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys AspLys 35 40 45 gac ggt aag tac agt cta aag gca aca gta gac aag att gag ctaaaa 192 Asp Gly Lys Tyr Ser Leu Lys Ala Thr Val Asp Lys Ile Glu Leu Lys50 55 60 gga act tct gat aaa gac aat ggt tct gga gtg ctt gaa ggt aca aaa240 Gly Thr Ser Asp Lys Asp Asn Gly Ser Gly Val Leu Glu Gly Thr Lys 6570 75 80 gat gac aaa agt aaa gca aaa tta aca att gct gac gat cta agt aaa288 Asp Asp Lys Ser Lys Ala Lys Leu Thr Ile Ala Asp Asp Leu Ser Lys 8590 95 acc aca ttc gaa ctt tta aaa gaa gat ggc aaa aca tta gtg tca aga336 Thr Thr Phe Glu Leu Leu Lys Glu Asp Gly Lys Thr Leu Val Ser Arg 100105 110 aaa gta agt tct aga gac aaa aca tca aca gat gaa atg ttc aat gaa384 Lys Val Ser Ser Arg Asp Lys Thr Ser Thr Asp Glu Met Phe Asn Glu 115120 125 aaa ggt gaa ttg tct gca aaa acc atg aca aga gaa aat gga acc aaa432 Lys Gly Glu Leu Ser Ala Lys Thr Met Thr Arg Glu Asn Gly Thr Lys 130135 140 ctt gaa tat aca gaa atg aaa agc gat gga acc gga aaa gct aaa gaa480 Leu Glu Tyr Thr Glu Met Lys Ser Asp Gly Thr Gly Lys Ala Lys Glu 145150 155 160 gtt tta aaa aag ttt act ctt gaa gga aaa gta gct aat gat aaagta 528 Val Leu Lys Lys Phe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys Val165 170 175 aca ttg gaa gta aaa gaa gga acc gtt act tta agt aag gaa attgca 576 Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu Ser Lys Glu Ile Ala180 185 190 aaa tct gga gaa gta aca gtt gct ctt aat gac act aac act actcag 624 Lys Ser Gly Glu Val Thr Val Ala Leu Asn Asp Thr Asn Thr Thr Gln195 200 205 gct act aaa aaa act ggc gca tgg gat tca aaa act tct act ttaaca 672 Ala Thr Lys Lys Thr Gly Ala Trp Asp Ser Lys Thr Ser Thr Leu Thr210 215 220 att agt gtt aac agc aaa aaa act aca caa ctt gtg ttt act aaacaa 720 Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu Val Phe Thr Lys Gln225 230 235 240 tac aca ata act gta aaa caa tac gac tcc gca ggt acc aattta gaa 768 Tyr Thr Ile Thr Val Lys Gln Tyr Asp Ser Ala Gly Thr Asn LeuGlu 245 250 255 ggc aca gca gtc gaa att aaa aca ctt gat gaa ctt aaa aacgct tta 816 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn AlaLeu 260 265 270 aaa taa 822 Lys * 11 273 PRT Borrelia burgdorferi 11 MetLys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Ala Ser Val 20 25 30Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45Asp Gly Lys Tyr Ser Leu Lys Ala Thr Val Asp Lys Ile Glu Leu Lys 50 55 60Gly Thr Ser Asp Lys Asp Asn Gly Ser Gly Val Leu Glu Gly Thr Lys 65 70 7580 Asp Asp Lys Ser Lys Ala Lys Leu Thr Ile Ala Asp Asp Leu Ser Lys 85 9095 Thr Thr Phe Glu Leu Leu Lys Glu Asp Gly Lys Thr Leu Val Ser Arg 100105 110 Lys Val Ser Ser Arg Asp Lys Thr Ser Thr Asp Glu Met Phe Asn Glu115 120 125 Lys Gly Glu Leu Ser Ala Lys Thr Met Thr Arg Glu Asn Gly ThrLys 130 135 140 Leu Glu Tyr Thr Glu Met Lys Ser Asp Gly Thr Gly Lys AlaLys Glu 145 150 155 160 Val Leu Lys Lys Phe Thr Leu Glu Gly Lys Val AlaAsn Asp Lys Val 165 170 175 Thr Leu Glu Val Lys Glu Gly Thr Val Thr LeuSer Lys Glu Ile Ala 180 185 190 Lys Ser Gly Glu Val Thr Val Ala Leu AsnAsp Thr Asn Thr Thr Gln 195 200 205 Ala Thr Lys Lys Thr Gly Ala Trp AspSer Lys Thr Ser Thr Leu Thr 210 215 220 Ile Ser Val Asn Ser Lys Lys ThrThr Gln Leu Val Phe Thr Lys Gln 225 230 235 240 Tyr Thr Ile Thr Val LysGln Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 Gly Thr Ala Val GluIle Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu 260 265 270 Lys 12 819 DNABorrelia burgdorferi CDS (1)...(819) 12 atg aaa aaa tat tta ttg gga ataggt cta ata tta gct tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile GlyLeu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc cttgac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu AspGlu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt cttgta agc aaa gaa aaa gac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu ValSer Lys Glu Lys Asp Lys 35 40 45 gac ggc aag tac agt cta atg gca aca gtagac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Ser Leu Met Ala Thr Val AspLys Leu Glu Leu Lys 50 55 60 gga aca tct gat aaa aac aat gga tct ggg gtgctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val LeuGlu Gly Val Lys 65 70 75 80 gct gac aaa agc aaa gta aaa tta aca gtt tctgac gat cta agc aca 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Val Ser AspAsp Leu Ser Thr 85 90 95 acc aca ctt gaa gtt tta aaa gaa gat ggc aaa acatta gtg tca aaa 336 Thr Thr Leu Glu Val Leu Lys Glu Asp Gly Lys Thr LeuVal Ser Lys 100 105 110 aaa aga act tct aaa gat aag tca tca aca gaa gaaaag ttc aat gaa 384 Lys Arg Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu LysPhe Asn Glu 115 120 125 aaa ggc gaa tta gtt gaa aaa ata atg gca aga gcaaac gga acc ata 432 Lys Gly Glu Leu Val Glu Lys Ile Met Ala Arg Ala AsnGly Thr Ile 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tcc ggaaaa gct aaa gaa 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly LysAla Lys Glu 145 150 155 160 act tta aaa gaa tat gtt ctt gaa gga act ctaact gct gaa aaa gca 528 Thr Leu Lys Glu Tyr Val Leu Glu Gly Thr Leu ThrAla Glu Lys Ala 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act ttaagt aag cac att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu SerLys His Ile Ser 180 185 190 aaa tct gga gaa gta aca gct gaa ctt aat gacact gac agt act caa 624 Lys Ser Gly Glu Val Thr Ala Glu Leu Asn Asp ThrAsp Ser Thr Gln 195 200 205 gct act aaa aaa act ggg aaa tgg gat gca ggcact tca act tta aca 672 Ala Thr Lys Lys Thr Gly Lys Trp Asp Ala Gly ThrSer Thr Leu Thr 210 215 220 att act gta aac aac aaa aaa act aaa gcc cttgta ttt aca aaa caa 720 Ile Thr Val Asn Asn Lys Lys Thr Lys Ala Leu ValPhe Thr Lys Gln 225 230 235 240 gac aca att aca tca caa aaa tac gac tcagca gga acc aac ttg gaa 768 Asp Thr Ile Thr Ser Gln Lys Tyr Asp Ser AlaGly Thr Asn Leu Glu 245 250 255 ggc aca gca gtc gaa att aaa aca ctt gatgaa ctt aaa aac gct tta 816 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp GluLeu Lys Asn Ala Leu 260 265 270 aga 819 Arg 13 273 PRT Borreliaburgdorferi 13 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala LeuIle Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn SerVal Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys GluLys Asp Lys 35 40 45 Asp Gly Lys Tyr Ser Leu Met Ala Thr Val Asp Lys LeuGlu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu GluGly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Val Ser AspAsp Leu Ser Thr 85 90 95 Thr Thr Leu Glu Val Leu Lys Glu Asp Gly Lys ThrLeu Val Ser Lys 100 105 110 Lys Arg Thr Ser Lys Asp Lys Ser Ser Thr GluGlu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Leu Val Glu Lys Ile Met AlaArg Ala Asn Gly Thr Ile 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser AspGly Ser Gly Lys Ala Lys Glu 145 150 155 160 Thr Leu Lys Glu Tyr Val LeuGlu Gly Thr Leu Thr Ala Glu Lys Ala 165 170 175 Thr Leu Val Val Lys GluGly Thr Val Thr Leu Ser Lys His Ile Ser 180 185 190 Lys Ser Gly Glu ValThr Ala Glu Leu Asn Asp Thr Asp Ser Thr Gln 195 200 205 Ala Thr Lys LysThr Gly Lys Trp Asp Ala Gly Thr Ser Thr Leu Thr 210 215 220 Ile Thr ValAsn Asn Lys Lys Thr Lys Ala Leu Val Phe Thr Lys Gln 225 230 235 240 AspThr Ile Thr Ser Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu 260 265270 Arg 14 21 DNA Artificial Sequence Oligonucleotide Primer 14gtctgcaaaa accatgacaa g 21 15 22 DNA Artificial Sequence OligonucleotidePrimer 15 gtcatcaaca gaagaaaaat tc 22 16 30 DNA Artificial SequenceOligonucleotide Primer 16 ccggatccat atgaaaaaat atttattggg 30 17 32 DNAArtificial Sequence Oligonucleotide Primer 17 ccgggatcca tatggctaagcaaaatgtta gc 32 18 18 DNA Artificial Sequence Oligonucleotide Primer 18gcgttcaagt actccaga 18 19 27 DNA Artificial Sequence OligonucleotidePrimer 19 gatatctaga tcttatttta aagcgtt 27 20 31 DNA Artificial SequenceOligonucleotide Primer 20 ggatccggtg accttttaaa gcgtttttaa t 31 21 825DNA Borrelia burgdorferi 21 atgaaaaaat atttattggg aataggtcta atattagccttaatagcatg taagcaaaat 60 gttagcagcc ttgatgaaaa aaatagcgtt tcagtagatttacctggtgg aatgacagtt 120 cttgtaagta aagaaaaaga caaagacggt aaatacagtctagaggcaac agtagacaag 180 cttgagctta aaggaacttc tgataaaaac aacggttctggaacacttga aggtgaaaaa 240 actgacaaaa gtaaagtaaa atcaacaatt gctgatgacctaagtcaaac taaatttgaa 300 attttcaaag aagatggcaa aacattagta tcaaaaaaagtaacccttaa agacaagtca 360 tcaacagaag aaaaattcaa cggaaagggt gaaacatctgaaaaaacaat agtaagagca 420 aatggaacca gacttgaata cacagacata aaaagcgatggatccggaaa agctaaagaa 480 gttttaaaag actttactct tgaaggaact ctagctgctgacggcaaaac aacattgaaa 540 gttacagaag gcactgttgt tttaagcaag aacattttaaaatccggaga aataacagct 600 gcacttgatg actctgacac tactcgggct actaaaaaaactggaaaatg ggattcaaag 660 acttccactt taacaattag tgtgaatagc caaaaaaccaaaaaccttgt attcacaaaa 720 gaagacacaa taacagtaca aagatacgac tcagcaggcaccaatctaga aggcaaagca 780 gtcgaaatta caacacttaa agaacttaaa aacgctttaaaataa 825 22 824 DNA Borrelia burgdorferi 22 atgaaaaaat atttattgggaataggtcta atattagcat taatagcatg taagcaaaat 60 gttagcagcc ttgatgaaaaaaatagcgtt tcagtagatt tacctggtgg aatgcaagtt 120 cttgtaagta aagaaaaagacaaagatggt aaatacagtc taatggcaac agtagacaag 180 cttgagctta aaggaacttctgataaaaac aacggttctg gaacacttga aggtgaaaaa 240 actgacaaaa gtaaagcaaaattaacaatt gctgaggatc taagtaaaac cacatttgaa 300 atcttcaaag aagatggcaaaacattagta tcaaaaaaag taacccttaa agacaagtca 360 tcaacagaag aaaaattcaacgcaaagggt gaagcatctg aaaaaacaat agtaagagca 420 aatggaacca gacttgaatacacagacata aaaagcgata aaaccggaaa agctaaagaa 480 gttttaaaag actttgctcttgaaggaact ctagctgctg acggcaaaac aacattaaaa 540 gttacagaag gcactgttgttttaagcaaa cacatttcaa actctggaga aataacagtt 600 gagcttaatg actctgacactactcaggct actaaaaaaa ctggaacatg ggattcaaag 660 acttccactt taacaattagtgtgaatagc cgaaaaacca aaaaccttgt attcacaaaa 720 gaagacacaa taacagtacaaaaatacgac tcagcaggca ccaatctaga aggcaaagca 780 gtcgaaatta caacgcttaaagaacttaaa gatgctttaa aata 824 23 821 DNA Borrelia burgdorferi 23atgaaaaaat atttattggg aataggtcta atattagcct taatagcatg caagcaaaat 60gttagcagcc ttgatgaaaa aaacagcgct tcagtagatt tgcctggtga gatgaaagtt 120cttgtaagta aagaaaaaga caaagacggt aagtacagtc taaaggcaac agtagacaag 180attgagctaa aaggaacttc tgataaagac aatggttctg gggtgcttga aggtacaaaa 240gatgacaaaa gtaaagcaaa attaacaatt gctgacgatc taggtaaaac cacattcgaa 300cttttcaaag aagatggcaa aacattagtg tcaagaaaag taagttctaa agacaaaaca 360tcaacagatg aaatgttcaa tgaaaaaggt gaattgtctg caaaaaccat gacaagagaa 420aatggaacca aacttgaata tacagaaatg aaaagcgatg gaaccggaaa agctaaagaa 480gttttaaaaa actttactct tgaaggaaaa gtagctaatg ataaagtaac attggaagta 540aaagaaggaa ccgttacttt aagtaaggaa attgcaaaat ctggagaagt aacagttgct 600cttaatgaca ctaacactac tcaggctact aaaaaaactg gcgcatggga ttcaaaaact 660tctactttaa caattagtgt taacagcaaa aaaactacac aacttgtgtt tactaaacaa 720gacacaataa ctgtacaaaa atacgactcc gcaggtacca atttagaagg cacagcagtc 780gaaattaaaa cacttgatga acttaaaaac gctttaaaat a 821 24 821 DNA Borreliaburgdorferi 24 atgaaaaaat atttattggg aataggtcta atattagcct taatagcatgcaagcaaaat 60 gttagcagcc ttgatgaaaa aaacagcgct tcagtagatt tgcctggtgagattaaagtt 120 cttgtaagta aagaaaaaga caaagacggt aagtacagtc taaaggcaacagtagacaag 180 attgagctaa aaggaacttc tgataaagac aatggttctg gagtgcttgaaggtacaaaa 240 gatgacaaaa gtaaagcaaa attaacaatt gctgacgatc taagtaaaaccacattcgaa 300 cttttcaaag aagatggcaa aacattagtg tcaagaaaag taagttctaaagacaaaaca 360 tcaacagatg aaatgttcaa tgaaaaaggt gaattgtctg caaaaaccatgacaagagaa 420 aatggaacca aacttgaata tacagaaatg aaaagcgatg gaaccggaaaagctaaagaa 480 gttttaaaaa actttactct tgaaggaaaa gtagctaatg ataaagtaacattggaagta 540 aaagaaggaa ccgttacttt aagtaaggaa attgcaaaat ctggagaagtaacagttgct 600 cttaatgaca ctaacactac tcaggctact aaaaaaactg gcgcatgggattcaaaaact 660 tctactttaa caattagtgt taacagtaaa aaaactacac aacttgtgtttactaaacaa 720 gacacaataa ctgtacaaaa atacgactcc gcaggtacca atttagaaggcacagcagtc 780 gaaattaaaa cacttgatga acttaaaaac gctttaaaat a 821 25 821DNA Borrelia burgdorferi 25 atgaaaaaat atttattggg aataggtcta atattagccttaatagcatg caagcaaaat 60 gttagcagcc ttgatgaaaa aaacagcgct tcagtagatttgcctggtga gatgaaagtt 120 cttgtaagta aagaaaaaga caaagacggt aagtacagtctaaaggcaac agtagacaag 180 attgagctaa aaggaacttc tgataaagac aatggttctggggtgcttga aggtacaaaa 240 gatgacaaaa gtaaagcaaa attaacaatt gctgacgatctaggtaaaac cacattcgaa 300 cttttcaaag aagatggcaa aacattagtg tcaagaaaagtaagttctaa agacaaaaca 360 tcaacagatg aaatgttcaa tgaaaaaggt gaattgtctgcaaaaaccat gacaagagaa 420 aatggaacca aacttgaata tacagaaatg aaaagcgatggaaccggaaa agctaaagaa 480 gttttaaaaa actttactct tgaaggaaaa gtagctaatgataaagtaac attggaagta 540 aaagaaggaa ccgttacttt aagtaaggaa attgcaaaatctggagaagt aacagttgct 600 cttaatgaca ctaacactac tcaggctact aaaaaaactggcgcatggga ttcaaaaact 660 tctactttaa caattagtgt taacagcaaa aaaactacacaacttgtgtt tactaaacaa 720 gacacaataa ctgtacaaaa atacgactcc gcaggtaccaatttagaagg cacagcagtc 780 gaaattaaaa cacttgatga acttaaaaac gctttaaaat a821 26 822 DNA Borrelia burgdorferi 26 atgaaaaaat atttattggg aataggtctaatattagcct taatagcatg caagcaaaat 60 gttagcagcc ttgatgaaaa aaacagcgcttcagtagatt tgcctggtga gatgaaagtt 120 cttgtaagta aagaaaaaga caaagacggtaagtacagtc taaaggcaac agtagacaag 180 attgagctaa aaggaacttc tgataaagacaatggttctg gagtgcttga aggtacaaaa 240 gatgacaaaa gtaaagcaaa attaacaattgctgacgatc taagtaaaac cacattcgaa 300 cttttcaaag aagatggcaa aacattagtgtcaagaaaag taagttctaa agacaaaaca 360 tcaacagatg aaatgttcaa tgaaaaaggtgaattgtctg caaaaaccat gacaagagaa 420 aatggaacca aacttgaata tacagaaatgaaaagcgatg gaaccggaaa agctaaagaa 480 gttttaaaaa actttactct tgaaggaaaagtagctaatg ataaagtaac attggaagta 540 aaagaaggaa ccgttacttt aagtaaggaaattgcaaaat ctggagaagt aacagttgct 600 cttaatgaca ctaacactac tcaggctactaaaaaaactg gcgcatggga ttcaaaaact 660 tctactttaa caattagtgt taacagcaaaaaaactacac aacttgtgtt tactaaacaa 720 gacacaataa ctgtacaaaa atacgactccgcaggtacca atttagaagg cacagcagtc 780 gaaattaaaa cacttgatga acttaaaaacgctttgaaat aa 822 27 38 DNA Artificial Sequence Oligonucleotide Primer27 aaagtagaag tttttgaatc ccattttcca gttttttt 38 28 825 DNA ArtificialSequence Chimeric nucleic acid 28 atg aaa aaa tat tta ttg gga ata ggtcta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly LeuIle Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gatgaa aaa aat agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp GluLys Asn Ser Val Ser Val 20 25 30 gat tta cct ggt gga atg aca gtt ctt gtaagt aaa gaa aaa gac aaa 144 Asp Leu Pro Gly Gly Met Thr Val Leu Val SerLys Glu Lys Asp Lys 35 40 45 gac ggt aaa tac agt cta gag gca aca gta gacaag ctt gag ctt aaa 192 Asp Gly Lys Tyr Ser Leu Glu Ala Thr Val Asp LysLeu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aac ggt tct gga aca cttgaa ggt gaa aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Thr Leu GluGly Glu Lys 65 70 75 80 act gac aaa agt aaa gta aaa tta aca att gct gatgac cta agt caa 288 Thr Asp Lys Ser Lys Val Lys Leu Thr Ile Ala Asp AspLeu Ser Gln 85 90 95 act aaa ttt gaa att ttc aaa gaa gat gcc aaa aca ttagta tca aaa 336 Thr Lys Phe Glu Ile Phe Lys Glu Asp Ala Lys Thr Leu ValSer Lys 100 105 110 aaa gta acc ctt aaa gac aag tca tca aca gaa gaa aaattc aac gaa 384 Lys Val Thr Leu Lys Asp Lys Ser Ser Thr Glu Glu Lys PheAsn Glu 115 120 125 aag ggt gaa aca tct gaa aaa aca ata gta aga gca aatgga acc aga 432 Lys Gly Glu Thr Ser Glu Lys Thr Ile Val Arg Ala Asn GlyThr Arg 130 135 140 ctt gaa tac aca gac ata aaa agc gat gga tcc gga aaagct aaa gaa 480 Leu Glu Tyr Thr Asp Ile Lys Ser Asp Gly Ser Gly Lys AlaLys Glu 145 150 155 160 gtt tta aaa gac ttt act ctt gaa gga act cta gctgct gac ggc aaa 528 Val Leu Lys Asp Phe Thr Leu Glu Gly Thr Leu Ala AlaAsp Gly Lys 165 170 175 aca aca ttg aaa gtt aca gaa ggc act gtt gtt ttaagc aag aac att 576 Thr Thr Leu Lys Val Thr Glu Gly Thr Val Val Leu SerLys Asn Ile 180 185 190 tta aaa tcc gga gaa ata aca gtt gca ctt gat gactct gac act act 624 Leu Lys Ser Gly Glu Ile Thr Val Ala Leu Asp Asp SerAsp Thr Thr 195 200 205 cag gct act aaa aaa act gga aaa tgg gat tca aaaact tct act tta 672 Gln Ala Thr Lys Lys Thr Gly Lys Trp Asp Ser Lys ThrSer Thr Leu 210 215 220 aca att agt gtt aac agc aaa aaa act aca caa cttgtg ttt act aaa 720 Thr Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu ValPhe Thr Lys 225 230 235 240 caa tac aca ata act gta aaa caa tac gac tccgca ggt acc aat tta 768 Gln Tyr Thr Ile Thr Val Lys Gln Tyr Asp Ser AlaGly Thr Asn Leu 245 250 255 gaa ggc aca gca gtc gaa att aaa aca ctt gatgaa ctt aaa aac gct 816 Glu Gly Thr Ala Val Glu Ile Lys Thr Leu Asp GluLeu Lys Asn Ala 260 265 270 tta aaa taa 825 Leu Lys * 29 274 PRTArtificial Sequence Chimeric protein 29 Met Lys Lys Tyr Leu Leu Gly IleGly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser SerLeu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Gly Met ThrVal Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 Asp Gly Lys Tyr Ser Leu GluAla Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn AsnGly Ser Gly Thr Leu Glu Gly Glu Lys 65 70 75 80 Thr Asp Lys Ser Lys ValLys Leu Thr Ile Ala Asp Asp Leu Ser Gln 85 90 95 Thr Lys Phe Glu Ile PheLys Glu Asp Ala Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Leu LysAsp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu ThrSer Glu Lys Thr Ile Val Arg Ala Asn Gly Thr Arg 130 135 140 Leu Glu TyrThr Asp Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 ValLeu Lys Asp Phe Thr Leu Glu Gly Thr Leu Ala Ala Asp Gly Lys 165 170 175Thr Thr Leu Lys Val Thr Glu Gly Thr Val Val Leu Ser Lys Asn Ile 180 185190 Leu Lys Ser Gly Glu Ile Thr Val Ala Leu Asp Asp Ser Asp Thr Thr 195200 205 Gln Ala Thr Lys Lys Thr Gly Lys Trp Asp Ser Lys Thr Ser Thr Leu210 215 220 Thr Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu Val Phe ThrLys 225 230 235 240 Gln Tyr Thr Ile Thr Val Lys Gln Tyr Asp Ser Ala GlyThr Asn Leu 245 250 255 Glu Gly Thr Ala Val Glu Ile Lys Thr Leu Asp GluLeu Lys Asn Ala 260 265 270 Leu Lys 30 822 DNA Artificial Sequence CDS(1)...(822) Chimeric nucleic acid 30 atg aaa aaa tat tta ttg gga ata ggtcta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly LeuIle Leu Ala Leu Ile Ala 1 5 10 15 tgc aag caa aat gtt agc agc ctt gatgaa aaa aac agc gct tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp GluLys Asn Ser Ala Ser Val 20 25 30 gat ttg cct ggt gag atg aaa gtt ctt gtaagt aaa gaa aaa gac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val SerLys Glu Lys Asp Lys 35 40 45 gac ggt aag tac agt cta aag gca aca gta gacaag att gag cta aaa 192 Asp Gly Lys Tyr Ser Leu Lys Ala Thr Val Asp LysIle Glu Leu Lys 50 55 60 gga act tct gat aaa gac aat ggt tct gga gtg cttgaa ggt aca aaa 240 Gly Thr Ser Asp Lys Asp Asn Gly Ser Gly Val Leu GluGly Thr Lys 65 70 75 80 gat gac aaa agt aaa gca aaa tta aca att gct gacgat cta agt aaa 288 Asp Asp Lys Ser Lys Ala Lys Leu Thr Ile Ala Asp AspLeu Ser Lys 85 90 95 acc aca ttc gaa ctt tta aaa gaa gat ggc aaa aca ttagtg tca aga 336 Thr Thr Phe Glu Leu Leu Lys Glu Asp Gly Lys Thr Leu ValSer Arg 100 105 110 aaa gta agt tct aga gac aaa aca tca aca gat gaa atgttc aat gaa 384 Lys Val Ser Ser Arg Asp Lys Thr Ser Thr Asp Glu Met PheAsn Glu 115 120 125 aaa ggt gaa ttg tct gca aaa acc atg aca aga gaa aatgga acc aaa 432 Lys Gly Glu Leu Ser Ala Lys Thr Met Thr Arg Glu Asn GlyThr Lys 130 135 140 ctt gaa tat aca gaa atg aaa agc gat gga acc gga aaagct aaa gaa 480 Leu Glu Tyr Thr Glu Met Lys Ser Asp Gly Thr Gly Lys AlaLys Glu 145 150 155 160 gtt tta aaa aag ttt act ctt gaa gga aaa gta gctaat gat aaa gta 528 Val Leu Lys Lys Phe Thr Leu Glu Gly Lys Val Ala AsnAsp Lys Val 165 170 175 aca ttg gaa gta aaa gaa gga acc gtt act tta agtaag gaa att gca 576 Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu Ser LysGlu Ile Ala 180 185 190 aaa tct gga gaa gta aca gtt gct ctt aat gac actaac act act cag 624 Lys Ser Gly Glu Val Thr Val Ala Leu Asn Asp Thr AsnThr Thr Gln 195 200 205 gct act aaa aaa act ggc gca tgg gat tca aaa acttct act tta aca 672 Ala Thr Lys Lys Thr Gly Ala Trp Asp Ser Lys Thr SerThr Leu Thr 210 215 220 att agt gtt aac agc aaa aaa act aca caa ctt gtgttt act aaa caa 720 Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu Val PheThr Lys Gln 225 230 235 240 tac aca ata act gta aaa caa tac gac tcc gcaggt acc aat tta gaa 768 Tyr Thr Ile Thr Val Lys Gln Tyr Asp Ser Ala GlyThr Asn Leu Glu 245 250 255 ggc aca gca gtc gaa att aaa aca ctt gat gaactt aaa aac gct tta 816 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu LeuLys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 31 273 PRT ArtificialSequence Chimeric protein 31 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu IleLeu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp GluLys Asn Ser Ala Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu ValSer Lys Glu Lys Asp Lys 35 40 45 Asp Gly Lys Tyr Ser Leu Lys Ala Thr ValAsp Lys Ile Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asp Asn Gly Ser GlyVal Leu Glu Gly Thr Lys 65 70 75 80 Asp Asp Lys Ser Lys Ala Lys Leu ThrIle Ala Asp Asp Leu Ser Lys 85 90 95 Thr Thr Phe Glu Leu Leu Lys Glu AspGly Lys Thr Leu Val Ser Arg 100 105 110 Lys Val Ser Ser Arg Asp Lys ThrSer Thr Asp Glu Met Phe Asn Glu 115 120 125 Lys Gly Glu Leu Ser Ala LysThr Met Thr Arg Glu Asn Gly Thr Lys 130 135 140 Leu Glu Tyr Thr Glu MetLys Ser Asp Gly Thr Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys LysPhe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys Val 165 170 175 Thr Leu GluVal Lys Glu Gly Thr Val Thr Leu Ser Lys Glu Ile Ala 180 185 190 Lys SerGly Glu Val Thr Val Ala Leu Asn Asp Thr Asn Thr Thr Gln 195 200 205 AlaThr Lys Lys Thr Gly Ala Trp Asp Ser Lys Thr Ser Thr Leu Thr 210 215 220Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu Val Phe Thr Lys Gln 225 230235 240 Tyr Thr Ile Thr Val Lys Gln Tyr Asp Ser Ala Gly Thr Asn Leu Glu245 250 255 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn AlaLeu 260 265 270 Lys 32 822 DNA Artificial Sequence Chimeric nucleic acid32 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 1015 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 AspLeu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gacggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp GlyLys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga acttct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr SerAsp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gacaaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp LysSer Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca cttgaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu GluVal Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta acttcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr SerLys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaagta tct gaa aaa ata ata aca aga gca gac gga acc aga 432 Lys Gly Glu ValSer Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 ctt gaa tacaca gga att aaa agc gat gga tct gga aaa gct aaa gag 480 Leu Glu Tyr ThrGly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt ttaaaa ggc tat gtt ctt gaa gga act cta act gct gaa aaa aca 528 Val Leu LysGly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 aca ttggtg gtt aaa gaa gga act gtt act tta agc aaa aat att tca 576 Thr Leu ValVal Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tctggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser GlyGlu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct actaaa aaa act gca gct tgg aat tca aaa act tcc act tta aca 672 Ala Thr LysLys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr 210 215 220 att agtgtg aat agc caa aaa acc aaa aac ctt gta ttc aca aaa gaa 720 Ile Ser ValAsn Ser Gln Lys Thr Lys Asn Leu Val Phe Thr Lys Glu 225 230 235 240 gacaca ata aca gta caa aaa tac gac tca gca ggc acc aat cta gaa 768 Asp ThrIle Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 ggcaaa gca gtc gaa att aca aca ctt aaa gaa ctt aaa aac gct tta 816 Gly LysAla Val Glu Ile Thr Thr Leu Lys Glu Leu Lys Asn Ala Leu 260 265 270 aaataa 822 Lys * 33 273 PRT Artificial Sequence Chimeric protein 33 Met LysLys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 CysLys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 AspLeu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 AspGly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 GlyThr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala LysGlu 145 150 155 160 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr AlaGlu Lys Thr 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu SerLys Asn Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn AspThr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn SerLys Thr Ser Thr Leu Thr 210 215 220 Ile Ser Val Asn Ser Gln Lys Thr LysAsn Leu Val Phe Thr Lys Glu 225 230 235 240 Asp Thr Ile Thr Val Gln LysTyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 Gly Lys Ala Val Glu IleThr Thr Leu Lys Glu Leu Lys Asn Ala Leu 260 265 270 Lys 34 819 DNAArtificial Sequence Chimeric nucleic acid 34 atg aaa aaa tat tta ttg ggaata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly IleGly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agcctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser LeuAsp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gttctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val LeuVal Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca acagta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr ValAsp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct ggagta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly ValLeu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca atttct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile SerAsp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaaaca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys ThrLeu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaagaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu GluLys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca agagca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg AlaAsp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tctgga aaa gct aaa gag 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser GlyLys Ala Lys Glu 145 150 155 160 gtt tta aaa ggc tat gtt ctt gaa gga actcta act gct gaa aaa aca 528 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr LeuThr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt acttta agc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr LeuSer Lys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aatgac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn AspThr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat gcaggc act tca act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ala GlyThr Ser Thr Leu Thr 210 215 220 att act gta aac aac aaa aaa act aaa gccctt gta ttt aca aaa caa 720 Ile Thr Val Asn Asn Lys Lys Thr Lys Ala LeuVal Phe Thr Lys Gln 225 230 235 240 gac aca att aca tca caa aaa tac gactca gca gga acc aac ttg gaa 768 Asp Thr Ile Thr Ser Gln Lys Tyr Asp SerAla Gly Thr Asn Leu Glu 245 250 255 ggc aca gca gtc gaa att aaa aca cttgat gaa ctt aaa aac gct tta 816 Gly Thr Ala Val Glu Ile Lys Thr Leu AspGlu Leu Lys Asn Ala Leu 260 265 270 aga 819 Arg 35 273 PRT ArtificialSequence Chimeric protein 35 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu IleLeu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp GluLys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu ValSer Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr ValAsp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser GlyVal Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu ThrIle Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu AspGly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys SerSer Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu LysIle Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly IleLys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys GlyTyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu ValVal Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys SerGly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 AlaThr Lys Lys Thr Ala Ala Trp Asn Ala Gly Thr Ser Thr Leu Thr 210 215 220Ile Thr Val Asn Asn Lys Lys Thr Lys Ala Leu Val Phe Thr Lys Gln 225 230235 240 Asp Thr Ile Thr Ser Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu245 250 255 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn AlaLeu 260 265 270 Arg 36 822 DNA Artificial Sequence Chimeric nucleic acid36 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 1015 tgt aag caa aat gtt agc agc ctt gat gaa aaa aat agc gtt tca gta 96Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30gat tta cct ggt gga atg aca gtt ctt gta agt aaa gaa aaa gac aaa 144 AspLeu Pro Gly Gly Met Thr Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 gacggt aaa tac agt cta gag gca aca gta gac aag ctt gag ctt aaa 192 Asp GlyLys Tyr Ser Leu Glu Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga acttct gat aaa aac aac ggt tct gga aca ctt gaa ggt gaa aaa 240 Gly Thr SerAsp Lys Asn Asn Gly Ser Gly Thr Leu Glu Gly Glu Lys 65 70 75 80 act gacaaa agt aaa gta aaa tta aca att gct gat gac cta agt caa 288 Thr Asp LysSer Lys Val Lys Leu Thr Ile Ala Asp Asp Leu Ser Gln 85 90 95 act aaa tttgaa att ttc aaa gaa gat gcc aaa aca tta gta tca aaa 336 Thr Lys Phe GluIle Phe Lys Glu Asp Ala Lys Thr Leu Val Ser Lys 100 105 110 aaa gta accctt aaa gac aag tca tca aca gaa gaa aaa ttc aac gaa 384 Lys Val Thr LeuLys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aag ggt gaaaca tct gaa aaa aca ata gta aga gca aat gga acc aga 432 Lys Gly Glu ThrSer Glu Lys Thr Ile Val Arg Ala Asn Gly Thr Arg 130 135 140 ctt gaa tacaca gac ata aaa agc gat gga tcc gga aaa gct aaa gaa 480 Leu Glu Tyr ThrAsp Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt ttaaaa gac ttt act ctt gaa gga act cta gct gct gac ggc aaa 528 Val Leu LysAsp Phe Thr Leu Glu Gly Thr Leu Ala Ala Asp Gly Lys 165 170 175 aca acattg aaa gtt aca gaa ggc act gtt gtt tta agc aag att tca 576 Thr Thr LeuLys Val Thr Glu Gly Thr Val Val Leu Ser Lys Ile Ser 180 185 190 aaa tctggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser GlyGlu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct actaaa aaa act gca gct tgg aat tca aaa act tcc act tta aca 672 Ala Thr LysLys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr 210 215 220 att agtgtg aat agc caa aaa acc aaa aac ctt gta ttc aca aaa gaa 720 Ile Ser ValAsn Ser Gln Lys Thr Lys Asn Leu Val Phe Thr Lys Glu 225 230 235 240 gacaca ata aca gta caa aaa tac gac tca gca ggc acc aat cta gaa 768 Asp ThrIle Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 ggcaaa gca gtc gaa att aca aca ctt aaa gaa ctt aaa aac gct tta 816 Gly LysAla Val Glu Ile Thr Thr Leu Lys Glu Leu Lys Asn Ala Leu 260 265 270 aaataa 822 Lys * 37 273 PRT Artificial Sequence Chimeric protein 37 Met LysLys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 CysLys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 AspLeu Pro Gly Gly Met Thr Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 AspGly Lys Tyr Ser Leu Glu Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 GlyThr Ser Asp Lys Asn Asn Gly Ser Gly Thr Leu Glu Gly Glu Lys 65 70 75 80Thr Asp Lys Ser Lys Val Lys Leu Thr Ile Ala Asp Asp Leu Ser Gln 85 90 95Thr Lys Phe Glu Ile Phe Lys Glu Asp Ala Lys Thr Leu Val Ser Lys 100 105110 Lys Val Thr Leu Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115120 125 Lys Gly Glu Thr Ser Glu Lys Thr Ile Val Arg Ala Asn Gly Thr Arg130 135 140 Leu Glu Tyr Thr Asp Ile Lys Ser Asp Gly Ser Gly Lys Ala LysGlu 145 150 155 160 Val Leu Lys Asp Phe Thr Leu Glu Gly Thr Leu Ala AlaAsp Gly Lys 165 170 175 Thr Thr Leu Lys Val Thr Glu Gly Thr Val Val LeuSer Lys Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn AspThr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn SerLys Thr Ser Thr Leu Thr 210 215 220 Ile Ser Val Asn Ser Gln Lys Thr LysAsn Leu Val Phe Thr Lys Glu 225 230 235 240 Asp Thr Ile Thr Val Gln LysTyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 Gly Lys Ala Val Glu IleThr Thr Leu Lys Glu Leu Lys Asn Ala Leu 260 265 270 Lys 38 822 DNAArtificial Sequence Chimeric nucleic acid 38 atg aaa aaa tat tta ttg ggaata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly IleGly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agcctt gat gaa aaa aat agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser LeuAsp Glu Lys Asn Ser Val Ser Val 20 25 30 gat tta cct ggt gga atg aca gttctt gta agt aaa gaa aaa gac aaa 144 Asp Leu Pro Gly Gly Met Thr Val LeuVal Ser Lys Glu Lys Asp Lys 35 40 45 gac ggt aaa tac agt cta gag gca acagta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Ser Leu Glu Ala Thr ValAsp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aac ggt tct ggaaca ctt gaa ggt gaa aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly ThrLeu Glu Gly Glu Lys 65 70 75 80 act gac aaa agt aaa gta aaa tta aca attgct gat gac cta agt caa 288 Thr Asp Lys Ser Lys Val Lys Leu Thr Ile AlaAsp Asp Leu Ser Gln 85 90 95 act aaa ttt gaa att ttc aaa gaa gat gcc aaaaca tta gta tca aaa 336 Thr Lys Phe Glu Ile Phe Lys Glu Asp Ala Lys ThrLeu Val Ser Lys 100 105 110 aaa gta acc ctt aaa gac aag tca tca aca gaagaa aaa ttc aac gaa 384 Lys Val Thr Leu Lys Asp Lys Ser Ser Thr Glu GluLys Phe Asn Glu 115 120 125 aag ggt gaa aca tct gaa aaa aca ata gta agagca aat gga acc aga 432 Lys Gly Glu Thr Ser Glu Lys Thr Ile Val Arg AlaAsn Gly Thr Arg 130 135 140 ctt gaa tac aca gac ata aaa agc gat gga tccgga aaa gct aaa gaa 480 Leu Glu Tyr Thr Asp Ile Lys Ser Asp Gly Ser GlyLys Ala Lys Glu 145 150 155 160 gtt tta aaa gac ttt act ctt gaa gga actcta gct gct gac ggc aaa 528 Val Leu Lys Asp Phe Thr Leu Glu Gly Thr LeuAla Ala Asp Gly Lys 165 170 175 aca aca ttg aaa gtt aca gaa ggc act gttgtt tta agc aag att tca 576 Thr Thr Leu Lys Val Thr Glu Gly Thr Val ValLeu Ser Lys Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aatgac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn AspThr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tcaaaa act tcc act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser LysThr Ser Thr Leu Thr 210 215 220 att agt gtg aat agc caa aaa acc aaa aacctt gta ttc aca aaa gaa 720 Ile Ser Val Asn Ser Gln Lys Thr Lys Asn LeuVal Phe Thr Lys Glu 225 230 235 240 gac aca ata aca gta caa aaa tac gactca gca ggc acc aat cta gaa 768 Asp Thr Ile Thr Val Gln Lys Tyr Asp SerAla Gly Thr Asn Leu Glu 245 250 255 ggc aaa gca gtc gaa att aca aca cttaaa gaa ctt aaa aac gct tta 816 Gly Lys Ala Val Glu Ile Thr Thr Leu LysGlu Leu Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 39 273 PRTArtificial Sequence Chimeric protein 39 Met Lys Lys Tyr Leu Leu Gly IleGly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser SerLeu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Gly Met ThrVal Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 Asp Gly Lys Tyr Ser Leu GluAla Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn AsnGly Ser Gly Thr Leu Glu Gly Glu Lys 65 70 75 80 Thr Asp Lys Ser Lys ValLys Leu Thr Ile Ala Asp Asp Leu Ser Gln 85 90 95 Thr Lys Phe Glu Ile PheLys Glu Asp Ala Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Leu LysAsp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu ThrSer Glu Lys Thr Ile Val Arg Ala Asn Gly Thr Arg 130 135 140 Leu Glu TyrThr Asp Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 ValLeu Lys Asp Phe Thr Leu Glu Gly Thr Leu Ala Ala Asp Gly Lys 165 170 175Thr Thr Leu Lys Val Thr Glu Gly Thr Val Val Leu Ser Lys Ile Ser 180 185190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr210 215 220 Ile Ser Val Asn Ser Gln Lys Thr Lys Asn Leu Val Phe Thr LysGlu 225 230 235 240 Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly ThrAsn Leu Glu 245 250 255 Gly Lys Ala Val Glu Ile Thr Thr Leu Lys Glu LeuLys Asn Ala Leu 260 265 270 Lys 40 822 DNA Artificial Sequence Chimericnucleic acid 40 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc ttaata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu IleAla 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtttca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val SerVal 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aacaaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 5055 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 6570 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 8590 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115120 125 aaa ggt gaa gta tct gaa aaa ata ata aca aga gca gac gga acc aga432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa gag480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145150 155 160 gtt tta aaa ggc tat gtt ctt gaa gga act cta act gct gaa aaaaca 528 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaa aat atttca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agtgct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala195 200 205 gct act aaa aaa act gca gct tgg aat gac agt act agc act ttaaca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Asp Ser Thr Ser Thr Leu Thr210 215 220 att agt gct gac agc aaa aaa act aaa gat ttg gtg ttc tta acagat 720 Ile Ser Ala Asp Ser Lys Lys Thr Lys Asp Leu Val Phe Leu Thr Asp225 230 235 240 ggt aca att aca gta caa caa tac aac aca gct gga acc agccta gaa 768 Gly Thr Ile Thr Val Gln Gln Tyr Asn Thr Ala Gly Thr Ser LeuGlu 245 250 255 gga tca gca agt gaa att aaa aat ctt tca gag ctt aaa aacgct tta 816 Gly Ser Ala Ser Glu Ile Lys Asn Leu Ser Glu Leu Lys Asn AlaLeu 260 265 270 aaa taa 822 Lys * 41 273 PRT Artificial SequenceChimeric protein 41 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu AlaLeu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys AsnSer Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser LysGlu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp LysLeu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val LeuGlu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile SerAsp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly LysThr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser ThrGlu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile IleThr Arg Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile Lys SerAsp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Gly Tyr ValLeu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val Val LysGlu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys Ser Gly GluVal Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr LysLys Thr Ala Ala Trp Asn Asp Ser Thr Ser Thr Leu Thr 210 215 220 Ile SerAla Asp Ser Lys Lys Thr Lys Asp Leu Val Phe Leu Thr Asp 225 230 235 240Gly Thr Ile Thr Val Gln Gln Tyr Asn Thr Ala Gly Thr Ser Leu Glu 245 250255 Gly Ser Ala Ser Glu Ile Lys Asn Leu Ser Glu Leu Lys Asn Ala Leu 260265 270 Lys 42 822 DNA Borrelia burgdorferi 42 atgaaaaaat atttattgggaataggtcta atattagcct taatagcatg taagcaaaat 60 gttagcagcc ttgacgagaaaaacagcgtt tcagtagatt tgcctggtga aatgaaagtt 120 cttgtaagca aagaaaaaaacaaagacggc aagtacgatc taattgcaac agtagacaag 180 cttgagctta aaggaacttctgataaaaac aatggatctg gagtacttga aggcgtaaaa 240 gctgacaaaa gtaaagtaaaattaacaatt tctgacgatc taggtcaaac cacacttgaa 300 gttttcaaag aagatggcaaaacactagta tcaaaaaaag taacttccaa agacaagtca 360 tcaacagaag aaaaattcaatgaaaaaggt gaagtatctg aaaaaataat aacaagagca 420 gacggaacca gacttgaatacacaggaatt aaaagcgatg gatctggaaa agctaaagag 480 gttttaaaag gctatgttcttgaaggaact ctaactgctg aaaaaacaac attggtggtt 540 aaagaaggaa ctgttactttaagcaaaaat atttcaaaat ctggggaagt ttcagttgaa 600 cttaatgaca ctgacagtagtgctgctact aaaaaaactg cagcttggaa ttcaggcact 660 tcaactttaa caattactgtaaacagtaaa aaaactaaag accttgtgtt tacaaaagaa 720 aacacaatta cagtacaacaatacgactca aatggcacca aattagaggg gtcagcagtt 780 gaaattacaa aacttgatgaaattaaaaac gctttaaaat aa 822 43 822 DNA Borrelia burgdorferi 43atgaaaaaat atttattggg aataggtcta atattagcct taatagcatg taagcaaaat 60gttagcagcc ttgacgagaa aaacagcgtt tcagtagatt tgcctggtga aatgaacgtt 120cttgtaagca aagaaaaaaa caaagacggc aagtacgatc taattgcaac agtagacaag 180cttgagctta aaggaacttc tgataaaaac aatggatctg gagtacttga aggcgtaaaa 240gctgacaaaa gtaaagtaaa attaacaatt tctgacgatc taggtcaaac cacacttgaa 300gttttcaaag aagatggcaa aacactagta tcaaaaaaag taacttccaa agacaagtca 360tcaacagaag aaaaattcaa tgaaaaaggt gaagtatctg aaaaaataat aacaagagca 420gacggaacca gacttgaata cacagaaatt aaaagcgatg gatctggaaa agctaaagag 480gttttaaaag gctatgttct tgaaggaact ctaactgctg aaaaaacaac attggtggtt 540aaagaaggaa ctgttacttt aagcaaaaat atttcaaaat ctggggaagt ttcagttgaa 600cttaatgaca ctgacagtag tgctgctact aaaaaaactg cagcttggaa ttcaggcact 660tcaactttaa caattactgt aaacagtaaa aaaactaaag accttgtgtt tacaaaagaa 720aacacaatta cagtacaaca atacgactca aatggcacca aattagaggg gtcagcagtt 780gaaattacaa aacttgatga aattaaaaac gctttaaaat aa 822 44 822 DNA Borreliaburgdorferi 44 atgaaaaaat atttattggg aataggtcta atattagcct taatagcatgtaagcaaaat 60 gttagcagcc ttgacgagaa aaacagcgtt tcagtagatt tgcctggtgaaatgaacgtt 120 cttgtaagca aagaaaaaaa caaagacggc aagtacgatc taattgcaacagtagacaag 180 cttgagctta aaggaacttc tgataaaaac aatggatctg gagtacttgaaggcgtaaaa 240 gctgacaaaa gtaaagtaaa attaacaatt tctgacgatc taggtcaaaccacacttgaa 300 gttttcaaag aagatggcaa aacactagta tcaaaaaaag taacttccaaagacaagtca 360 tcaacagaag aaaaattcaa tgaaaaaggt gaagtatctg aaaaaataataacaagagca 420 gacggaacca gacttgaata cacagaaatt aaaagcgatg gatctggaaaagctaaagag 480 gttttaaaaa gctatgttct tgaaggaact ttaactgctg aaaaaacaacattggtggtt 540 aaagaaggaa ctgttacttt aagcaaaaat atttcaaaat ctggggaagtttcagttgaa 600 cttaatgaca ctgacagtag tgctgctact aaaaaaactg cagcttggaattcaggcact 660 tcaactttaa caattactgt aaacagtaaa aaaactaaag accttgtgtttacaaaagaa 720 aacacaatta cagtacaaca atacgactca aatggcacca aattagaggggtcagcagtt 780 gaaattacaa aacttgatga aattaaaaac gctttaaaat aa 822 45 821DNA Borrelia burgdorferi 45 atgaaaaaat atttattggg aataggtcta atattagccttaatagcatg taagcaaaat 60 gttagcagcc ttgatgagaa aaacagcgtt tcagtagatttacctggtga aatgaaagtt 120 cttgtaagca aagaaaaaga caaagatggt aaatacagtctaatggcaac agtagacaag 180 ctagagctta aaggaacttc tgataaaagc aacggttctggaacacttga aggtgaaaaa 240 tctgacaaaa gtaaagcaaa attaacaatt tctgaagatctaagtaaaac cacatttgaa 300 attttcaaag aagatggcaa aacattagta tcaaaaaaagtaaattctaa agataagtca 360 tcaatagaag aaaaattcaa cgcaaaaggt gaattatctgaaaaaacaat actaagagca 420 aacggaacca ggcttgaata cacagaaata aaaagcgatggaaccggaaa agctaaagaa 480 gctttaaaag actttgctct tgaaggaact ctagctgccgacaaaacaac attgaaagtt 540 acagaaggca ctgttgtttt aagcaaacac attccaaactctggagaaat aacagttgag 600 cttaatgact ctaactctac tcaggctact aaaaaaactggaaaatggga ttcaaatact 660 tccactttaa caattagtgt gaatagcaaa aaaactaaaaacattgtatt tacaaaagaa 720 gacacaataa cagtacaaaa atacgactca gcaggcaccaatctagaagg caacgcagtc 780 gaaattaaaa cacttgatga acttaaaaac gctttaaaat a821 46 821 DNA Borrelia burgdorferi 46 atgaaaaaat atttattggg aataggtctaatattagcct taatagcatg taagcaaaat 60 gttagcagcc ttgatgaaaa aaatagcgtttcagtagatt tacctggtgg aatgaaagtt 120 cttgtaagta aagaaaaaga caaagatggtaaatacagtc taatggcaac agtagaaaag 180 cttgagctta aaggaacttc tgataaaaacaacggttctg gaacacttga aggtgaaaaa 240 actgacaaaa gtaaagtaaa attaacaattgctgaggatc taagtaaaac cacatttgaa 300 atcttcaaag aagatggcaa aacattagtatcgaaaaaag taacccttaa agacaagtca 360 tcaacagaag aaaaattcaa cgaaaagggtgaaatatctg aaaaaacaat agtaagagca 420 aatggaacca gacttgaata cacagacataaaaagcgata aaaccggaaa agctaaagaa 480 gttttaaaag actttactct tgaaggaactctagctgctg acggcaaaac aacattgaaa 540 gttacagagg gcactgttac tttaagcaagaacatttcaa aatccggaga aataacagtt 600 gcacttgatg acactgactc tagcggcaataaaaaatccg gaacatggga ttcaggtact 660 tctactttaa caattagtaa aaacagacaaaaaactaaac aacttgtatt cacaaaagaa 720 gacacaataa cagtacaaaa ctacgactcagcaggcacca atctagaagg caaagcagtc 780 gaaattacaa cacttaaaga acttaaaaacgctttaaaat a 821 47 825 DNA Artificial Sequence Chimeric nucleic acid 47atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 MetLys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15tgt aag caa aat gtt agc agc ctt gat gaa aaa aat agc gtt tca gta 96 CysLys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gattta cct ggt gga atg aca gtt ctt gta agt aaa gaa aaa gac aaa 144 Asp LeuPro Gly Gly Met Thr Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 gac ggtaaa tac agt cta gag gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly LysTyr Ser Leu Glu Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tctgat aaa aac aac ggt tct gga aca ctt gaa ggt gaa aaa 240 Gly Thr Ser AspLys Asn Asn Gly Ser Gly Thr Leu Glu Gly Glu Lys 65 70 75 80 act gac aaaagt aaa gta aaa tta aca att gct gat gac cta agt caa 288 Thr Asp Lys SerLys Val Lys Leu Thr Ile Ala Asp Asp Leu Ser Gln 85 90 95 act aaa ttt gaaatt ttc aaa gaa gat gcc aaa aca tta gta tca aaa 336 Thr Lys Phe Glu IlePhe Lys Glu Asp Ala Lys Thr Leu Val Ser Lys 100 105 110 aaa gta acc cttaaa gac aag tca tca aca gaa gaa aaa ttc aac gaa 384 Lys Val Thr Leu LysAsp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aag ggt gaa acatct gaa aaa aca ata gta aga gca aat gga acc aga 432 Lys Gly Glu Thr SerGlu Lys Thr Ile Val Arg Ala Asn Gly Thr Arg 130 135 140 ctt gaa tac acagac ata aaa agc gat gga tcc gga aaa gct aaa gaa 480 Leu Glu Tyr Thr AspIle Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaagac ttt act ctt gaa gga act cta gct gct gac ggc aaa 528 Val Leu Lys AspPhe Thr Leu Glu Gly Thr Leu Ala Ala Asp Gly Lys 165 170 175 aca aca ttgaaa gtt aca gaa ggc act gtt gtt tta agc aag aac att 576 Thr Thr Leu LysVal Thr Glu Gly Thr Val Val Leu Ser Lys Asn Ile 180 185 190 tta aaa tccgga gaa ata aca gtt gca ctt gat gac tct gac act act 624 Leu Lys Ser GlyGlu Ile Thr Val Ala Leu Asp Asp Ser Asp Thr Thr 195 200 205 cag gct actaaa aaa act gga aaa tgg gat tca aat act tcc act tta 672 Gln Ala Thr LysLys Thr Gly Lys Trp Asp Ser Asn Thr Ser Thr Leu 210 215 220 aca att agtgtg aat agc aaa aaa act aaa aac att gta ttt aca aaa 720 Thr Ile Ser ValAsn Ser Lys Lys Thr Lys Asn Ile Val Phe Thr Lys 225 230 235 240 gaa gacaca ata aca gta caa aaa tac gac tca gca ggc acc aat cta 768 Glu Asp ThrIle Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu 245 250 255 gaa ggcaac gca gtc gaa att aaa aca ctt gat gaa ctt aaa aac gct 816 Glu Gly AsnAla Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala 260 265 270 tta aaatag 825 Leu Lys * 48 274 PRT Artificial Sequence Chimeric protein 48 MetLys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30Asp Leu Pro Gly Gly Met Thr Val Leu Val Ser Lys Glu Lys Asp Lys 35 40 45Asp Gly Lys Tyr Ser Leu Glu Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Thr Leu Glu Gly Glu Lys 65 70 7580 Thr Asp Lys Ser Lys Val Lys Leu Thr Ile Ala Asp Asp Leu Ser Gln 85 9095 Thr Lys Phe Glu Ile Phe Lys Glu Asp Ala Lys Thr Leu Val Ser Lys 100105 110 Lys Val Thr Leu Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu115 120 125 Lys Gly Glu Thr Ser Glu Lys Thr Ile Val Arg Ala Asn Gly ThrArg 130 135 140 Leu Glu Tyr Thr Asp Ile Lys Ser Asp Gly Ser Gly Lys AlaLys Glu 145 150 155 160 Val Leu Lys Asp Phe Thr Leu Glu Gly Thr Leu AlaAla Asp Gly Lys 165 170 175 Thr Thr Leu Lys Val Thr Glu Gly Thr Val ValLeu Ser Lys Asn Ile 180 185 190 Leu Lys Ser Gly Glu Ile Thr Val Ala LeuAsp Asp Ser Asp Thr Thr 195 200 205 Gln Ala Thr Lys Lys Thr Gly Lys TrpAsp Ser Asn Thr Ser Thr Leu 210 215 220 Thr Ile Ser Val Asn Ser Lys LysThr Lys Asn Ile Val Phe Thr Lys 225 230 235 240 Glu Asp Thr Ile Thr ValGln Lys Tyr Asp Ser Ala Gly Thr Asn Leu 245 250 255 Glu Gly Asn Ala ValGlu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala 260 265 270 Leu Lys 49 822DNA Artificial Sequence Chimeric nucleic acid 49 atg aaa aaa tat tta ttggga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu GlyIle Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgc aag caa aat gtt agcagc ctt gat gaa aaa aac agc gct tca gta 96 Cys Lys Gln Asn Val Ser SerLeu Asp Glu Lys Asn Ser Ala Ser Val 20 25 30 gat ttg cct ggt gag atg aaagtt ctt gta agt aaa gaa aaa gac aaa 144 Asp Leu Pro Gly Glu Met Lys ValLeu Val Ser Lys Glu Lys Asp Lys 35 40 45 gac ggt aag tac agt cta aag gcaaca gta gac aag att gag cta aaa 192 Asp Gly Lys Tyr Ser Leu Lys Ala ThrVal Asp Lys Ile Glu Leu Lys 50 55 60 gga act tct gat aaa gac aat ggt tctgga gtg ctt gaa ggt aca aaa 240 Gly Thr Ser Asp Lys Asp Asn Gly Ser GlyVal Leu Glu Gly Thr Lys 65 70 75 80 gat gac aaa agt aaa gca aaa tta acaatt gct gac gat cta agt aaa 288 Asp Asp Lys Ser Lys Ala Lys Leu Thr IleAla Asp Asp Leu Ser Lys 85 90 95 acc aca ttc gaa ctt tta aaa gaa gat ggcaaa aca tta gtg tca aga 336 Thr Thr Phe Glu Leu Leu Lys Glu Asp Gly LysThr Leu Val Ser Arg 100 105 110 aaa gta agt tct aga gac aaa aca tca acagat gaa atg ttc aat gaa 384 Lys Val Ser Ser Arg Asp Lys Thr Ser Thr AspGlu Met Phe Asn Glu 115 120 125 aaa ggt gaa ttg tct gca aaa acc atg acaaga gaa aat gga acc aaa 432 Lys Gly Glu Leu Ser Ala Lys Thr Met Thr ArgGlu Asn Gly Thr Lys 130 135 140 ctt gaa tat aca gaa atg aaa agc gat ggaacc gga aaa gct aaa gaa 480 Leu Glu Tyr Thr Glu Met Lys Ser Asp Gly ThrGly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa aag ttt act ctt gaa ggaaaa gta gct aat gat aaa gta 528 Val Leu Lys Lys Phe Thr Leu Glu Gly LysVal Ala Asn Asp Lys Val 165 170 175 aca ttg gaa gta aaa gaa gga acc gttact tta agt aag gaa att gca 576 Thr Leu Glu Val Lys Glu Gly Thr Val ThrLeu Ser Lys Glu Ile Ala 180 185 190 aaa tct gga gaa gta aca gtt gct cttaat gac act aac act act cag 624 Lys Ser Gly Glu Val Thr Val Ala Leu AsnAsp Thr Asn Thr Thr Gln 195 200 205 gct act aaa aaa act ggc gca tgg gattca aaa act tct act tta aca 672 Ala Thr Lys Lys Thr Gly Ala Trp Asp SerLys Thr Ser Thr Leu Thr 210 215 220 att agt gtt aac agc aaa aaa act acacaa ctt gtg ttt act aaa caa 720 Ile Ser Val Asn Ser Lys Lys Thr Thr GlnLeu Val Phe Thr Lys Gln 225 230 235 240 gac aca ata act gta caa aaa tacgac tcc gca ggt acc aat tta gaa 768 Asp Thr Ile Thr Val Gln Lys Tyr AspSer Ala Gly Thr Asn Leu Glu 245 250 255 ggt aca gca gtc gaa att aaa acactt gat gaa ctt aaa aac gct tta 816 Gly Thr Ala Val Glu Ile Lys Thr LeuAsp Glu Leu Lys Asn Ala Leu 260 265 270 aaa tag 822 Lys * 50 273 PRTArtificial Sequence Chimeric protein 50 Met Lys Lys Tyr Leu Leu Gly IleGly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser SerLeu Asp Glu Lys Asn Ser Ala Ser Val 20 25 30 Asp Leu Pro Gly Glu Met LysVal Leu Val Ser Lys Glu Lys Asp Lys 35 40 45 Asp Gly Lys Tyr Ser Leu LysAla Thr Val Asp Lys Ile Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asp AsnGly Ser Gly Val Leu Glu Gly Thr Lys 65 70 75 80 Asp Asp Lys Ser Lys AlaLys Leu Thr Ile Ala Asp Asp Leu Ser Lys 85 90 95 Thr Thr Phe Glu Leu LeuLys Glu Asp Gly Lys Thr Leu Val Ser Arg 100 105 110 Lys Val Ser Ser ArgAsp Lys Thr Ser Thr Asp Glu Met Phe Asn Glu 115 120 125 Lys Gly Glu LeuSer Ala Lys Thr Met Thr Arg Glu Asn Gly Thr Lys 130 135 140 Leu Glu TyrThr Glu Met Lys Ser Asp Gly Thr Gly Lys Ala Lys Glu 145 150 155 160 ValLeu Lys Lys Phe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys Val 165 170 175Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu Ser Lys Glu Ile Ala 180 185190 Lys Ser Gly Glu Val Thr Val Ala Leu Asn Asp Thr Asn Thr Thr Gln 195200 205 Ala Thr Lys Lys Thr Gly Ala Trp Asp Ser Lys Thr Ser Thr Leu Thr210 215 220 Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu Val Phe Thr LysGln 225 230 235 240 Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly ThrAsn Leu Glu 245 250 255 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu LeuLys Asn Ala Leu 260 265 270 Lys 51 822 DNA Artificial Sequence Chimericnucleic acid 51 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc ttaata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu IleAla 1 5 10 15 tgc aag caa aat gtt agc agc ctt gat gaa aaa aac agc gcttca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Ala SerVal 20 25 30 gat ttg cct ggt gag atg aaa gtt ctt gta agt aaa gaa aaa gacaaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asp Lys35 40 45 gac ggt aag tac agt cta aag gca aca gta gac aag att gag cta aaa192 Asp Gly Lys Tyr Ser Leu Lys Ala Thr Val Asp Lys Ile Glu Leu Lys 5055 60 gga act tct gat aaa gac aat ggt tct gga gtg ctt gaa ggt aca aaa240 Gly Thr Ser Asp Lys Asp Asn Gly Ser Gly Val Leu Glu Gly Thr Lys 6570 75 80 gat gac aaa agt aaa gca aaa tta aca att gct gac gat cta agt aaa288 Asp Asp Lys Ser Lys Ala Lys Leu Thr Ile Ala Asp Asp Leu Ser Lys 8590 95 acc aca ttc gaa ctt tta aaa gaa gat ggc aaa aca tta gtg tca aga336 Thr Thr Phe Glu Leu Leu Lys Glu Asp Gly Lys Thr Leu Val Ser Arg 100105 110 aaa gta agt tct aga gac aaa aca tca aca gat gaa atg ttc aat gaa384 Lys Val Ser Ser Arg Asp Lys Thr Ser Thr Asp Glu Met Phe Asn Glu 115120 125 aaa ggt gaa ttg tct gca aaa acc atg aca aga gaa aat gga acc aaa432 Lys Gly Glu Leu Ser Ala Lys Thr Met Thr Arg Glu Asn Gly Thr Lys 130135 140 ctt gaa tat aca gaa atg aaa agc gat gga acc gga aaa gct aaa gaa480 Leu Glu Tyr Thr Glu Met Lys Ser Asp Gly Thr Gly Lys Ala Lys Glu 145150 155 160 gtt tta aaa aag ttt act ctt gaa gga aaa gta gct aat gat aaagta 528 Val Leu Lys Lys Phe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys Val165 170 175 aca ttg gaa gta aaa gaa gga acc gtt act tta agt aag gaa atttca 576 Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu Ser Lys Glu Ile Ser180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agtgct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala195 200 205 gct act aaa aaa act gca gct tgg aat tca aaa act tcc act ttaaca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr210 215 220 att agt gtg aat agc caa aaa acc aaa aac ctt gta ttc aca aaagaa 720 Ile Ser Val Asn Ser Gln Lys Thr Lys Asn Leu Val Phe Thr Lys Glu225 230 235 240 gac aca ata aca gta caa aaa tac gac tca gca ggc acc aatcta gaa 768 Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn LeuGlu 245 250 255 ggc aaa gca gtc gaa att aca aca ctt aaa gaa ctt aaa aacgct tta 816 Gly Lys Ala Val Glu Ile Thr Thr Leu Lys Glu Leu Lys Asn AlaLeu 260 265 270 aaa taa 822 Lys * 52 273 PRT Artificial SequenceChimeric protein 52 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu AlaLeu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys AsnSer Ala Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser LysGlu Lys Asp Lys 35 40 45 Asp Gly Lys Tyr Ser Leu Lys Ala Thr Val Asp LysIle Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asp Asn Gly Ser Gly Val LeuGlu Gly Thr Lys 65 70 75 80 Asp Asp Lys Ser Lys Ala Lys Leu Thr Ile AlaAsp Asp Leu Ser Lys 85 90 95 Thr Thr Phe Glu Leu Leu Lys Glu Asp Gly LysThr Leu Val Ser Arg 100 105 110 Lys Val Ser Ser Arg Asp Lys Thr Ser ThrAsp Glu Met Phe Asn Glu 115 120 125 Lys Gly Glu Leu Ser Ala Lys Thr MetThr Arg Glu Asn Gly Thr Lys 130 135 140 Leu Glu Tyr Thr Glu Met Lys SerAsp Gly Thr Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Lys Phe ThrLeu Glu Gly Lys Val Ala Asn Asp Lys Val 165 170 175 Thr Leu Glu Val LysGlu Gly Thr Val Thr Leu Ser Lys Glu Ile Ser 180 185 190 Lys Ser Gly GluVal Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr LysLys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr 210 215 220 Ile SerVal Asn Ser Gln Lys Thr Lys Asn Leu Val Phe Thr Lys Glu 225 230 235 240Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250255 Gly Lys Ala Val Glu Ile Thr Thr Leu Lys Glu Leu Lys Asn Ala Leu 260265 270 Lys 53 822 DNA Artificial Sequence Chimeric nucleic acid 53 atgaaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met LysLys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgtaag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys LysGln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttgcct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu ProGly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aagtac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys TyrAsp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gataaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp LysAsn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agtaaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser LysVal Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gttttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val PheLys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaagac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys AspLys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tctgaa aaa ata ata aca aga gca aat gga acc aaa 432 Lys Gly Glu Val Ser GluLys Ile Ile Thr Arg Ala Asn Gly Thr Lys 130 135 140 ctt gaa tat aca gaaatg aaa agc gat gga acc gga aaa gct aaa gaa 480 Leu Glu Tyr Thr Glu MetLys Ser Asp Gly Thr Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa aagttt act ctt gaa gga aaa gta gct aat gat aaa gta 528 Val Leu Lys Lys PheThr Leu Glu Gly Lys Val Ala Asn Asp Lys Val 165 170 175 aca ttg gaa gtaaaa gaa gga acc gtt act tta agt aag gaa att tca 576 Thr Leu Glu Val LysGlu Gly Thr Val Thr Leu Ser Lys Glu Ile Ser 180 185 190 aaa tct ggg gaagtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu ValSer Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaaact gca gct tgg aat tca aaa act tcc act tta aca 672 Ala Thr Lys Lys ThrAla Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr 210 215 220 att agt gtg aatagc caa aaa acc aaa aac ctt gta ttc aca aaa gaa 720 Ile Ser Val Asn SerGln Lys Thr Lys Asn Leu Val Phe Thr Lys Glu 225 230 235 240 gac aca ataaca gta caa aaa tac gac tca gca ggc acc aat cta gaa 768 Asp Thr Ile ThrVal Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 ggc aaa gcagtc gaa att aca aca ctt aaa gaa ctt aaa aac gct tta 816 Gly Lys Ala ValGlu Ile Thr Thr Leu Lys Glu Leu Lys Asn Ala Leu 260 265 270 aaa taa 822Lys * 54 273 PRT Artificial Sequence Chimeric protein 54 Met Lys Lys TyrLeu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys GlnAsn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu ProGly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly LysTyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr SerAsp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala AspLys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr ThrLeu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 LysVal Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asn Gly Thr Lys 130 135140 Leu Glu Tyr Thr Glu Met Lys Ser Asp Gly Thr Gly Lys Ala Lys Glu 145150 155 160 Val Leu Lys Lys Phe Thr Leu Glu Gly Lys Val Ala Asn Asp LysVal 165 170 175 Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu Ser Lys GluIle Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr AspSer Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys ThrSer Thr Leu Thr 210 215 220 Ile Ser Val Asn Ser Gln Lys Thr Lys Asn LeuVal Phe Thr Lys Glu 225 230 235 240 Asp Thr Ile Thr Val Gln Lys Tyr AspSer Ala Gly Thr Asn Leu Glu 245 250 255 Gly Lys Ala Val Glu Ile Thr ThrLeu Lys Glu Leu Lys Asn Ala Leu 260 265 270 Lys 55 1362 DNA ArtificialSequence Chimeric nucleic acid 55 atg gct tgt aat aat tca ggg aaa gatggg aat aca tct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly Lys Asp GlyAsn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tct gtt aaa ggg cct aatctt aca gaa ata aat aaa aaa 96 Ala Asp Glu Ser Val Lys Gly Pro Asn LeuThr Glu Ile Asn Lys Lys 20 25 30 att acg gat tct aat gcg gtt tta ctt gctgtg aaa gag gtt gaa gcg 144 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala ValLys Glu Val Glu Ala 35 40 45 ttg ctg tca tct ata gat gaa att gct gct aaagct att ggt aaa aaa 192 Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys AlaIle Gly Lys Lys 50 55 60 ata cac caa aat aat ggt ttg gat acc gaa aat aatcac aat gga tca 240 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Asn Asn HisAsn Gly Ser 65 70 75 80 ttg tta gcg gga gct tat gca ata tca acc cta ataaaa caa aaa tta 288 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile LysGln Lys Leu 85 90 95 gat gga ttg aaa aat gaa gga tta aag gaa aaa att gatgcg gct aag 336 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp AlaAla Lys 100 105 110 aaa tgt tct gaa aca ttt act aat aaa tta aaa gaa aaacac aca gat 384 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys HisThr Asp 115 120 125 ctt ggt aaa gaa ggt gtt act gat gct gat gca aaa gaagcc att tta 432 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu AlaIle Leu 130 135 140 aaa gca aat ggt act aaa act aaa ggt gct gaa gaa cttgga aaa tta 480 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu GlyLys Leu 145 150 155 160 ttt gaa tca gta gag gtc ttg tca aaa gca gct aaagag atg ctt gct 528 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys GluMet Leu Ala 165 170 175 aat tca gtt aaa gag ctt aca agc cct gtt gtg gcagaa agt cca aaa 576 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala GluSer Pro Lys 180 185 190 aaa cct tcc atg gcc aag caa aat gtt agc agc cttgac gag aaa aac 624 Lys Pro Ser Met Ala Lys Gln Asn Val Ser Ser Leu AspGlu Lys Asn 195 200 205 agc gtt tca gta gat ttg cct ggt gaa atg aaa gttctt gta agc aaa 672 Ser Val Ser Val Asp Leu Pro Gly Glu Met Lys Val LeuVal Ser Lys 210 215 220 gaa aaa aac aaa gac ggc aag tac gat cta att gcaaca gta gac aag 720 Glu Lys Asn Lys Asp Gly Lys Tyr Asp Leu Ile Ala ThrVal Asp Lys 225 230 235 240 ctt gag ctt aaa gga act tct gat aaa aac aatgga tct gga gta ctt 768 Leu Glu Leu Lys Gly Thr Ser Asp Lys Asn Asn GlySer Gly Val Leu 245 250 255 gaa ggc gta aaa gct gac aaa agt aaa gta aaatta aca att tct gac 816 Glu Gly Val Lys Ala Asp Lys Ser Lys Val Lys LeuThr Ile Ser Asp 260 265 270 gat cta ggt caa acc aca ctt gaa gtt ttc aaagaa gat ggc aaa aca 864 Asp Leu Gly Gln Thr Thr Leu Glu Val Phe Lys GluAsp Gly Lys Thr 275 280 285 cta gta tca aaa aaa gta act tcc aaa gac aagtca tca aca gaa gaa 912 Leu Val Ser Lys Lys Val Thr Ser Lys Asp Lys SerSer Thr Glu Glu 290 295 300 aaa ttc aat gaa aaa ggt gaa gta tct gaa aaaata ata aca aga gca 960 Lys Phe Asn Glu Lys Gly Glu Val Ser Glu Lys IleIle Thr Arg Ala 305 310 315 320 gac gga acc aga ctt gaa tac aca gga attaaa agc gat gga tct gga 1008 Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile LysSer Asp Gly Ser Gly 325 330 335 aaa gct aaa gag gtt tta aaa ggc tat gttctt gaa gga act cta act 1056 Lys Ala Lys Glu Val Leu Lys Gly Tyr Val LeuGlu Gly Thr Leu Thr 340 345 350 gct gaa aaa aca aca ttg gtg gtt aaa gaagga act gtt act tta agc 1104 Ala Glu Lys Thr Thr Leu Val Val Lys Glu GlyThr Val Thr Leu Ser 355 360 365 aaa aat att tca aaa tct ggg gaa gtt tcagtt gaa ctt aat gac act 1152 Lys Asn Ile Ser Lys Ser Gly Glu Val Ser ValGlu Leu Asn Asp Thr 370 375 380 gac agt agt gct gct act aaa aaa act gcagct tgg aat tca ggc act 1200 Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala AlaTrp Asn Ser Gly Thr 385 390 395 400 tca act tta aca att act gta aac agtaaa aaa act aaa gac ctt gtg 1248 Ser Thr Leu Thr Ile Thr Val Asn Ser LysLys Thr Lys Asp Leu Val 405 410 415 ttt aca aaa gaa aac aca att aca gtacaa caa tac gac tca aat ggc 1296 Phe Thr Lys Glu Asn Thr Ile Thr Val GlnGln Tyr Asp Ser Asn Gly 420 425 430 acc aaa tta gag ggg tca gca gtt gaaatt aca aaa ctt gat gaa att 1344 Thr Lys Leu Glu Gly Ser Ala Val Glu IleThr Lys Leu Asp Glu Ile 435 440 445 aaa aac gct tta aaa taa 1362 Lys AsnAla Leu Lys * 450 56 453 PRT Artificial Sequence Chimeric protein 56 MetAla Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60Ile His Gln Asn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 7580 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 9095 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100105 110 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp115 120 125 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala IleLeu 130 135 140 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu GlyLys Leu 145 150 155 160 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala LysGlu Met Leu Ala 165 170 175 Asn Ser Val Lys Glu Leu Thr Ser Pro Val ValAla Glu Ser Pro Lys 180 185 190 Lys Pro Ser Met Ala Lys Gln Asn Val SerSer Leu Asp Glu Lys Asn 195 200 205 Ser Val Ser Val Asp Leu Pro Gly GluMet Lys Val Leu Val Ser Lys 210 215 220 Glu Lys Asn Lys Asp Gly Lys TyrAsp Leu Ile Ala Thr Val Asp Lys 225 230 235 240 Leu Glu Leu Lys Gly ThrSer Asp Lys Asn Asn Gly Ser Gly Val Leu 245 250 255 Glu Gly Val Lys AlaAsp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp 260 265 270 Asp Leu Gly GlnThr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr 275 280 285 Leu Val SerLys Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu 290 295 300 Lys PheAsn Glu Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala 305 310 315 320Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly 325 330335 Lys Ala Lys Glu Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr 340345 350 Ala Glu Lys Thr Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser355 360 365 Lys Asn Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn AspThr 370 375 380 Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn SerGly Thr 385 390 395 400 Ser Thr Leu Thr Ile Thr Val Asn Ser Lys Lys ThrLys Asp Leu Val 405 410 415 Phe Thr Lys Glu Asn Thr Ile Thr Val Gln GlnTyr Asp Ser Asn Gly 420 425 430 Thr Lys Leu Glu Gly Ser Ala Val Glu IleThr Lys Leu Asp Glu Ile 435 440 445 Lys Asn Ala Leu Lys 450 57 1353 DNAArtificial Sequence Chimeric nucleic acid 57 atg gct tgt aat aat tca gggaaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly LysAsp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tct gtt aaa gggcct aat ctt aca gaa ata aat aaa aaa 96 Ala Asp Glu Ser Val Lys Gly ProAsn Leu Thr Glu Ile Asn Lys Lys 20 25 30 att acg gat tct aat gcg gtt ttactt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Asp Ser Asn Ala Val Leu LeuAla Val Lys Glu Val Glu Ala 35 40 45 ttg ctg tca tct ata gat gaa att gctgct aaa gct att ggt aaa aaa 192 Leu Leu Ser Ser Ile Asp Glu Ile Ala AlaLys Ala Ile Gly Lys Lys 50 55 60 ata cac caa aat aat ggt ttg gat acc gaaaat aat cac aat gga tca 240 Ile His Gln Asn Asn Gly Leu Asp Thr Glu AsnAsn His Asn Gly Ser 65 70 75 80 ttg tta gcg gga gct tat gca ata tca acccta ata aaa caa aaa tta 288 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr LeuIle Lys Gln Lys Leu 85 90 95 gat gga ttg aaa aat gaa gga tta aag gaa aaaatt gat gcg gct aag 336 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys IleAsp Ala Ala Lys 100 105 110 aaa tgt tct gaa aca ttt act aat aaa tta aaagaa aaa cac aca gat 384 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys GluLys His Thr Asp 115 120 125 ctt ggt aaa gaa ggt gtt act gat gct gat gcaaaa gaa gcc att tta 432 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala LysGlu Ala Ile Leu 130 135 140 aaa gca aat ggt act aaa act aaa ggt gct gaagaa ctt gga aaa tta 480 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu GluLeu Gly Lys Leu 145 150 155 160 ttt gaa tca gta gag gtc ttg tca aaa gcagct aaa gag atg ctt gct 528 Phe Glu Ser Val Glu Val Leu Ser Lys Ala AlaLys Glu Met Leu Ala 165 170 175 aat tca gtt aaa gag ctt aca agc cct gttgtg gca gaa agt cca gcc 576 Asn Ser Val Lys Glu Leu Thr Ser Pro Val ValAla Glu Ser Pro Ala 180 185 190 atg gcc aag caa aat gtt agc agc ctt gacgag aaa aac agc gtt tca 624 Met Ala Lys Gln Asn Val Ser Ser Leu Asp GluLys Asn Ser Val Ser 195 200 205 gta gat ttg cct ggt gaa atg aaa gtt cttgta agc aaa gaa aaa aac 672 Val Asp Leu Pro Gly Glu Met Lys Val Leu ValSer Lys Glu Lys Asn 210 215 220 aaa gac ggc aag tac gat cta att gca acagta gac aag ctt gag ctt 720 Lys Asp Gly Lys Tyr Asp Leu Ile Ala Thr ValAsp Lys Leu Glu Leu 225 230 235 240 aaa gga act tct gat aaa aac aat ggatct gga gta ctt gaa ggc gta 768 Lys Gly Thr Ser Asp Lys Asn Asn Gly SerGly Val Leu Glu Gly Val 245 250 255 aaa gct gac aaa agt aaa gta aaa ttaaca att tct gac gat cta ggt 816 Lys Ala Asp Lys Ser Lys Val Lys Leu ThrIle Ser Asp Asp Leu Gly 260 265 270 caa acc aca ctt gaa gtt ttc aaa gaagat ggc aaa aca cta gta tca 864 Gln Thr Thr Leu Glu Val Phe Lys Glu AspGly Lys Thr Leu Val Ser 275 280 285 aaa aaa gta act tcc aaa gac aag tcatca aca gaa gaa aaa ttc aat 912 Lys Lys Val Thr Ser Lys Asp Lys Ser SerThr Glu Glu Lys Phe Asn 290 295 300 gaa aaa ggt gaa gta tct gaa aaa ataata aca aga gca gac gga acc 960 Glu Lys Gly Glu Val Ser Glu Lys Ile IleThr Arg Ala Asp Gly Thr 305 310 315 320 aga ctt gaa tac aca gga att aaaagc gat gga tct gga aaa gct aaa 1008 Arg Leu Glu Tyr Thr Gly Ile Lys SerAsp Gly Ser Gly Lys Ala Lys 325 330 335 gag gtt tta aaa ggc tat gtt cttgaa gga act cta act gct gaa aaa 1056 Glu Val Leu Lys Gly Tyr Val Leu GluGly Thr Leu Thr Ala Glu Lys 340 345 350 aca aca ttg gtg gtt aaa gaa ggaact gtt act tta agc aaa aat att 1104 Thr Thr Leu Val Val Lys Glu Gly ThrVal Thr Leu Ser Lys Asn Ile 355 360 365 tca aaa tct ggg gaa gtt tca gttgaa ctt aat gac act gac agt agt 1152 Ser Lys Ser Gly Glu Val Ser Val GluLeu Asn Asp Thr Asp Ser Ser 370 375 380 gct gct act aaa aaa act gca gcttgg aat tca ggc act tca act tta 1200 Ala Ala Thr Lys Lys Thr Ala Ala TrpAsn Ser Gly Thr Ser Thr Leu 385 390 395 400 aca att act gta aac agt aaaaaa act aaa gac ctt gtg ttt aca aaa 1248 Thr Ile Thr Val Asn Ser Lys LysThr Lys Asp Leu Val Phe Thr Lys 405 410 415 gaa aac aca att aca gta caacaa tac gac tca aat ggc acc aaa tta 1296 Glu Asn Thr Ile Thr Val Gln GlnTyr Asp Ser Asn Gly Thr Lys Leu 420 425 430 gag ggg tca gca gtt gaa attaca aaa ctt gat gaa att aaa aac gct 1344 Glu Gly Ser Ala Val Glu Ile ThrLys Leu Asp Glu Ile Lys Asn Ala 435 440 445 tta aaa taa 1353 Leu Lys *450 58 450 PRT Artificial Sequence Chimeric protein 58 Met Ala Cys AsnAsn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp GluSer Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 Ile Thr AspSer Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu SerSer Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 Ile His GlnAsn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 Leu LeuAla Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 Asp GlyLeu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 LysCys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135140 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145150 155 160 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met LeuAla 165 170 175 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu SerPro Ala 180 185 190 Met Ala Lys Gln Asn Val Ser Ser Leu Asp Glu Lys AsnSer Val Ser 195 200 205 Val Asp Leu Pro Gly Glu Met Lys Val Leu Val SerLys Glu Lys Asn 210 215 220 Lys Asp Gly Lys Tyr Asp Leu Ile Ala Thr ValAsp Lys Leu Glu Leu 225 230 235 240 Lys Gly Thr Ser Asp Lys Asn Asn GlySer Gly Val Leu Glu Gly Val 245 250 255 Lys Ala Asp Lys Ser Lys Val LysLeu Thr Ile Ser Asp Asp Leu Gly 260 265 270 Gln Thr Thr Leu Glu Val PheLys Glu Asp Gly Lys Thr Leu Val Ser 275 280 285 Lys Lys Val Thr Ser LysAsp Lys Ser Ser Thr Glu Glu Lys Phe Asn 290 295 300 Glu Lys Gly Glu ValSer Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr 305 310 315 320 Arg Leu GluTyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys 325 330 335 Glu ValLeu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys 340 345 350 ThrThr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile 355 360 365Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser 370 375380 Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu 385390 395 400 Thr Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe ThrLys 405 410 415 Glu Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly ThrLys Leu 420 425 430 Glu Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu IleLys Asn Ala 435 440 445 Leu Lys 450 59 1341 DNA Artificial SequenceChimeric nucleic acid 59 atg gct tgt aat aat tca ggg aaa gat ggg aat acatct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr SerAla Asn Ser 1 5 10 15 gct gat gag tct gtt aaa ggg cct aat ctt aca gaaata agt aaa aaa 96 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu IleSer Lys Lys 20 25 30 att acg gat tct aat gcg gtt tta ctt gct gtg aaa gaggtt gaa gcg 144 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu ValGlu Ala 35 40 45 ttg ctg tca tct ata gat gag ctt gct aaa gct att ggt aaaaaa ata 192 Leu Leu Ser Ser Ile Asp Glu Leu Ala Lys Ala Ile Gly Lys LysIle 50 55 60 aaa aac gat ggt agt tta gat aat gaa gca aat cgc aac gag tcattg 240 Lys Asn Asp Gly Ser Leu Asp Asn Glu Ala Asn Arg Asn Glu Ser Leu65 70 75 80 tta gca gga gct tat aca ata tca acc tta ata aca caa aaa ttaagt 288 Leu Ala Gly Ala Tyr Thr Ile Ser Thr Leu Ile Thr Gln Lys Leu Ser85 90 95 aaa tta aac gga tca gaa ggt tta aag gaa aag att gcc gca gct aag336 Lys Leu Asn Gly Ser Glu Gly Leu Lys Glu Lys Ile Ala Ala Ala Lys 100105 110 aaa tgc tct gaa gag ttt agt act aaa cta aaa gat aat cat gca cag384 Lys Cys Ser Glu Glu Phe Ser Thr Lys Leu Lys Asp Asn His Ala Gln 115120 125 ctt ggt ata cag ggc gtt act gat gaa aat gca aaa aaa gct att tta432 Leu Gly Ile Gln Gly Val Thr Asp Glu Asn Ala Lys Lys Ala Ile Leu 130135 140 aaa gca aat gca gcg ggt aaa gat aag ggc gtt gaa gaa ctt gaa aag480 Lys Ala Asn Ala Ala Gly Lys Asp Lys Gly Val Glu Glu Leu Glu Lys 145150 155 160 ttg tcc gga tca tta gaa agc tta tca aaa gca gct aaa gag atgctt 528 Leu Ser Gly Ser Leu Glu Ser Leu Ser Lys Ala Ala Lys Glu Met Leu165 170 175 gct aat tca gtt aaa gag ctt aca agc cct gtt gtc cat ggc aagcaa 576 Ala Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val His Gly Lys Gln180 185 190 aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta gat ttgcct 624 Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val Asp Leu Pro195 200 205 ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa gac ggcaag 672 Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys Asp Gly Lys210 215 220 tac gat cta att gca aca gta gac aag ctt gag ctt aaa gga acttct 720 Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys Gly Thr Ser225 230 235 240 gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa gctgac aaa 768 Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys Ala AspLys 245 250 255 agt aaa gta aaa tta aca att tct gac gat cta ggt caa accaca ctt 816 Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln Thr ThrLeu 260 265 270 gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa aaagta act 864 Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys Lys ValThr 275 280 285 tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa aaaggt gaa 912 Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu Lys GlyGlu 290 295 300 gta tct gaa aaa ata ata aca aga gca gac gga acc aga cttgaa tac 960 Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg Leu GluTyr 305 310 315 320 aca gga att aaa agc gat gga tct gga aaa gct aaa gaggtt tta aaa 1008 Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu ValLeu Lys 325 330 335 ggc tat gtt ctt gaa gga act cta act gct gaa aaa acaaca ttg gtg 1056 Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr ThrLeu Val 340 345 350 gtt aaa gaa gga act gtt act tta agc aaa aat att tcaaaa tct ggg 1104 Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser LysSer Gly 355 360 365 gaa gtt tca gtt gaa ctt aat gac act gac agt agt gctgct act aaa 1152 Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala AlaThr Lys 370 375 380 aaa act gca gct tgg aat tca ggc act tca act tta acaatt act gta 1200 Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr IleThr Val 385 390 395 400 aac agt aaa aaa act aaa gac ctt gtg ttt aca aaagaa aac aca att 1248 Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys GluAsn Thr Ile 405 410 415 aca gta caa caa tac gac tca aat ggc acc aaa ttagag ggg tca gca 1296 Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu GluGly Ser Ala 420 425 430 gtt gaa att aca aaa ctt gat gaa att aaa aac gcttta aaa taa 1341 Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala LeuLys * 435 440 445 60 446 PRT Artificial Sequence Chimeric protein 60 MetAla Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45Leu Leu Ser Ser Ile Asp Glu Leu Ala Lys Ala Ile Gly Lys Lys Ile 50 55 60Lys Asn Asp Gly Ser Leu Asp Asn Glu Ala Asn Arg Asn Glu Ser Leu 65 70 7580 Leu Ala Gly Ala Tyr Thr Ile Ser Thr Leu Ile Thr Gln Lys Leu Ser 85 9095 Lys Leu Asn Gly Ser Glu Gly Leu Lys Glu Lys Ile Ala Ala Ala Lys 100105 110 Lys Cys Ser Glu Glu Phe Ser Thr Lys Leu Lys Asp Asn His Ala Gln115 120 125 Leu Gly Ile Gln Gly Val Thr Asp Glu Asn Ala Lys Lys Ala IleLeu 130 135 140 Lys Ala Asn Ala Ala Gly Lys Asp Lys Gly Val Glu Glu LeuGlu Lys 145 150 155 160 Leu Ser Gly Ser Leu Glu Ser Leu Ser Lys Ala AlaLys Glu Met Leu 165 170 175 Ala Asn Ser Val Lys Glu Leu Thr Ser Pro ValVal His Gly Lys Gln 180 185 190 Asn Val Ser Ser Leu Asp Glu Lys Asn SerVal Ser Val Asp Leu Pro 195 200 205 Gly Glu Met Lys Val Leu Val Ser LysGlu Lys Asn Lys Asp Gly Lys 210 215 220 Tyr Asp Leu Ile Ala Thr Val AspLys Leu Glu Leu Lys Gly Thr Ser 225 230 235 240 Asp Lys Asn Asn Gly SerGly Val Leu Glu Gly Val Lys Ala Asp Lys 245 250 255 Ser Lys Val Lys LeuThr Ile Ser Asp Asp Leu Gly Gln Thr Thr Leu 260 265 270 Glu Val Phe LysGlu Asp Gly Lys Thr Leu Val Ser Lys Lys Val Thr 275 280 285 Ser Lys AspLys Ser Ser Thr Glu Glu Lys Phe Asn Glu Lys Gly Glu 290 295 300 Val SerGlu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg Leu Glu Tyr 305 310 315 320Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu Val Leu Lys 325 330335 Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr Thr Leu Val 340345 350 Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser Lys Ser Gly355 360 365 Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala Ala ThrLys 370 375 380 Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr IleThr Val 385 390 395 400 Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr LysGlu Asn Thr Ile 405 410 415 Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr LysLeu Glu Gly Ser Ala 420 425 430 Val Glu Ile Thr Lys Leu Asp Glu Ile LysAsn Ala Leu Lys 435 440 445 61 1362 DNA Artificial Sequence Chimericnucleic acid 61 atg gct tgt aat aat tca ggg aaa gat ggg aat aca tct gcaaat tct 48 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala AsnSer 1 5 10 15 gct gat gag tct gtt aaa ggg cct aat ctt aca gaa ata aataaa aaa 96 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn LysLys 20 25 30 att acg gat tct aat gcg gtt tta ctt gct gtg aaa gag gtt gaagcg 144 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala35 40 45 ttg ctg tca tct ata gat gaa att gct gct aaa gct att ggt aaa aaa192 Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 5055 60 ata cac caa aat aat ggt ttg gat acc gaa aat aat cac aat gga tca240 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 6570 75 80 ttg tta gcg gga gct tat gca ata tca acc cta ata aaa caa aaa tta288 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 8590 95 gat gga ttg aaa aat gaa gga tta aag gaa aaa att gat gcg gct aag336 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100105 110 aaa tgt tct gaa aca ttt act aat aaa tta aaa gaa aaa cac aca gat384 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115120 125 ctt ggt aaa gaa ggt gtt act gat gct gat gca aaa gaa gcc att tta432 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130135 140 aaa gca aat ggt act aaa act aaa ggt gct gaa gaa ctt gga aaa tta480 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145150 155 160 ttt gaa tca gta gag gtc ttg tca aaa gca gct aaa gag atg cttgct 528 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala165 170 175 aat tca gtt aaa gag ctt aca agc cct gtt gtg gca gaa agt ccaaaa 576 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys180 185 190 aaa cct tcc atg gcc aag caa aat gtt agc agc ctt gac gag aaaaac 624 Lys Pro Ser Met Ala Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn195 200 205 agc gtt tca gta gat ttg cct ggt gaa atg aaa gtt ctt gta agcaaa 672 Ser Val Ser Val Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys210 215 220 gaa aaa aac aaa gac ggc aag tac gat cta att gca aca gta gacaag 720 Glu Lys Asn Lys Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys225 230 235 240 ctt gag ctt aaa gga act tct gat aaa aac aat gga tct ggagta ctt 768 Leu Glu Leu Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly ValLeu 245 250 255 gaa ggc gta aaa gct gac aaa agt aaa gta aaa tta aca atttct gac 816 Glu Gly Val Lys Ala Asp Lys Ser Lys Val Lys Leu Thr Ile SerAsp 260 265 270 gat cta ggt caa acc aca ctt gaa gtt ttc aaa gaa gat ggcaaa aca 864 Asp Leu Gly Gln Thr Thr Leu Glu Val Phe Lys Glu Asp Gly LysThr 275 280 285 cta gta tca aaa aaa gta act tcc aaa gac aag tca tca acagaa gaa 912 Leu Val Ser Lys Lys Val Thr Ser Lys Asp Lys Ser Ser Thr GluGlu 290 295 300 aaa ttc aat gaa aaa ggt gaa gta tct gaa aaa ata ata acaaga gca 960 Lys Phe Asn Glu Lys Gly Glu Val Ser Glu Lys Ile Ile Thr ArgAla 305 310 315 320 gac gga acc aga ctt gaa tac aca gga att aaa agc gatgga tct gga 1008 Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp GlySer Gly 325 330 335 aaa gct aaa gag gtt tta aaa ggc tat gtt ctt gaa ggaact cta act 1056 Lys Ala Lys Glu Val Leu Lys Gly Tyr Val Leu Glu Gly ThrLeu Thr 340 345 350 gct gaa aaa aca aca ttg gtg gtt aaa gaa gga act gttact tta agc 1104 Ala Glu Lys Thr Thr Leu Val Val Lys Glu Gly Thr Val ThrLeu Ser 355 360 365 aaa aat att tca aaa tct ggg gaa gtt tca gtt gaa cttaat gac act 1152 Lys Asn Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu AsnAsp Thr 370 375 380 gac agt agt gct gct act aaa aaa act gca gct tgg aattca aaa act 1200 Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn SerLys Thr 385 390 395 400 tcc act tta aca att agt gtg aat agc caa aaa accaaa aac ctt gta 1248 Ser Thr Leu Thr Ile Ser Val Asn Ser Gln Lys Thr LysAsn Leu Val 405 410 415 ttc aca aaa gaa gac aca ata aca gta caa aaa tacgac tca gca ggc 1296 Phe Thr Lys Glu Asp Thr Ile Thr Val Gln Lys Tyr AspSer Ala Gly 420 425 430 acc aat cta gaa ggc aaa gca gtc gaa att aca acactt aaa gaa ctt 1344 Thr Asn Leu Glu Gly Lys Ala Val Glu Ile Thr Thr LeuLys Glu Leu 435 440 445 aaa aac gct tta aaa taa 1362 Lys Asn Ala LeuLys * 450 62 453 PRT Artificial Sequence Chimeric protein 62 Met Ala CysAsn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala AspGlu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 Ile ThrAsp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu LeuSer Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 Ile HisGln Asn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 LeuLeu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 AspGly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120125 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130135 140 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu145 150 155 160 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu MetLeu Ala 165 170 175 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala GluSer Pro Lys 180 185 190 Lys Pro Ser Met Ala Lys Gln Asn Val Ser Ser LeuAsp Glu Lys Asn 195 200 205 Ser Val Ser Val Asp Leu Pro Gly Glu Met LysVal Leu Val Ser Lys 210 215 220 Glu Lys Asn Lys Asp Gly Lys Tyr Asp LeuIle Ala Thr Val Asp Lys 225 230 235 240 Leu Glu Leu Lys Gly Thr Ser AspLys Asn Asn Gly Ser Gly Val Leu 245 250 255 Glu Gly Val Lys Ala Asp LysSer Lys Val Lys Leu Thr Ile Ser Asp 260 265 270 Asp Leu Gly Gln Thr ThrLeu Glu Val Phe Lys Glu Asp Gly Lys Thr 275 280 285 Leu Val Ser Lys LysVal Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu 290 295 300 Lys Phe Asn GluLys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala 305 310 315 320 Asp GlyThr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly 325 330 335 LysAla Lys Glu Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr 340 345 350Ala Glu Lys Thr Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser 355 360365 Lys Asn Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr 370375 380 Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys Thr385 390 395 400 Ser Thr Leu Thr Ile Ser Val Asn Ser Gln Lys Thr Lys AsnLeu Val 405 410 415 Phe Thr Lys Glu Asp Thr Ile Thr Val Gln Lys Tyr AspSer Ala Gly 420 425 430 Thr Asn Leu Glu Gly Lys Ala Val Glu Ile Thr ThrLeu Lys Glu Leu 435 440 445 Lys Asn Ala Leu Lys 450 63 1341 DNAArtificial Sequence Chimeric nucleic acid 63 atg gct tgt aat aat tca gggaaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly LysAsp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tct gtt aaa gggcct aat ctt aca gaa ata agt aaa aaa 96 Ala Asp Glu Ser Val Lys Gly ProAsn Leu Thr Glu Ile Ser Lys Lys 20 25 30 att acg gat tct aat gcg gtt ttactt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Asp Ser Asn Ala Val Leu LeuAla Val Lys Glu Val Glu Ala 35 40 45 ttg ctg tca tct ata gat gag ctt gctaaa gct att ggt aaa aaa ata 192 Leu Leu Ser Ser Ile Asp Glu Leu Ala LysAla Ile Gly Lys Lys Ile 50 55 60 aaa aac gat ggt agt tta gat aat gaa gcaaat cgc aac gag tca ttg 240 Lys Asn Asp Gly Ser Leu Asp Asn Glu Ala AsnArg Asn Glu Ser Leu 65 70 75 80 tta gca gga gct tat aca ata tca acc ttaata aca caa aaa tta agt 288 Leu Ala Gly Ala Tyr Thr Ile Ser Thr Leu IleThr Gln Lys Leu Ser 85 90 95 aaa tta aac gga tca gaa ggt tta aag gaa aagatt gcc gca gct aag 336 Lys Leu Asn Gly Ser Glu Gly Leu Lys Glu Lys IleAla Ala Ala Lys 100 105 110 aaa tgc tct gaa gag ttt agt act aaa cta aaagat aat cat gca cag 384 Lys Cys Ser Glu Glu Phe Ser Thr Lys Leu Lys AspAsn His Ala Gln 115 120 125 ctt ggt ata cag ggc gtt act gat gaa aat gcaaaa aaa gct att tta 432 Leu Gly Ile Gln Gly Val Thr Asp Glu Asn Ala LysLys Ala Ile Leu 130 135 140 aaa gca aat gca gcg ggt aaa gat aag ggc gttgaa gaa ctt gaa aag 480 Lys Ala Asn Ala Ala Gly Lys Asp Lys Gly Val GluGlu Leu Glu Lys 145 150 155 160 ttg tcc gga tca tta gaa agc tta tca aaagca gct aaa gag atg ctt 528 Leu Ser Gly Ser Leu Glu Ser Leu Ser Lys AlaAla Lys Glu Met Leu 165 170 175 gct aat tca gtt aaa gag ctt aca agc cctgtt gtc cat ggc aag caa 576 Ala Asn Ser Val Lys Glu Leu Thr Ser Pro ValVal His Gly Lys Gln 180 185 190 aat gtt agc agc ctt gac gag aaa aac agcgtt tca gta gat ttg cct 624 Asn Val Ser Ser Leu Asp Glu Lys Asn Ser ValSer Val Asp Leu Pro 195 200 205 ggt gaa atg aaa gtt ctt gta agc aaa gaaaaa aac aaa gac ggc aag 672 Gly Glu Met Lys Val Leu Val Ser Lys Glu LysAsn Lys Asp Gly Lys 210 215 220 tac gat cta att gca aca gta gac aag cttgag ctt aaa gga act tct 720 Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu GluLeu Lys Gly Thr Ser 225 230 235 240 gat aaa aac aat gga tct gga gta cttgaa ggc gta aaa gct gac aaa 768 Asp Lys Asn Asn Gly Ser Gly Val Leu GluGly Val Lys Ala Asp Lys 245 250 255 agt aaa gta aaa tta aca att tct gacgat cta ggt caa acc aca ctt 816 Ser Lys Val Lys Leu Thr Ile Ser Asp AspLeu Gly Gln Thr Thr Leu 260 265 270 gaa gtt ttc aaa gaa gat ggc aaa acacta gta tca aaa aaa gta act 864 Glu Val Phe Lys Glu Asp Gly Lys Thr LeuVal Ser Lys Lys Val Thr 275 280 285 tcc aaa gac aag tca tca aca gaa gaaaaa ttc aat gaa aaa ggt gaa 912 Ser Lys Asp Lys Ser Ser Thr Glu Glu LysPhe Asn Glu Lys Gly Glu 290 295 300 gta tct gaa aaa ata ata aca aga gcagac gga acc aga ctt gaa tac 960 Val Ser Glu Lys Ile Ile Thr Arg Ala AspGly Thr Arg Leu Glu Tyr 305 310 315 320 aca gga att aaa agc gat gga tctgga aaa gct aaa gag gtt tta aaa 1008 Thr Gly Ile Lys Ser Asp Gly Ser GlyLys Ala Lys Glu Val Leu Lys 325 330 335 ggc tat gtt ctt gaa gga act ctaact gct gaa aaa aca aca ttg gtg 1056 Gly Tyr Val Leu Glu Gly Thr Leu ThrAla Glu Lys Thr Thr Leu Val 340 345 350 gtt aaa gaa gga act gtt act ttaagc aaa aat att tca aaa tct ggg 1104 Val Lys Glu Gly Thr Val Thr Leu SerLys Asn Ile Ser Lys Ser Gly 355 360 365 gaa gtt tca gtt gaa ctt aat gacact gac agt agt gct gct act aaa 1152 Glu Val Ser Val Glu Leu Asn Asp ThrAsp Ser Ser Ala Ala Thr Lys 370 375 380 aaa act gca gct tgg aat tca aaaact tcc act tta aca att agt gtg 1200 Lys Thr Ala Ala Trp Asn Ser Lys ThrSer Thr Leu Thr Ile Ser Val 385 390 395 400 aat agc caa aaa acc aaa aacctt gta ttc aca aaa gaa gac aca ata 1248 Asn Ser Gln Lys Thr Lys Asn LeuVal Phe Thr Lys Glu Asp Thr Ile 405 410 415 aca gta caa aaa tac gac tcagca ggc acc aat cta gaa ggc aaa gca 1296 Thr Val Gln Lys Tyr Asp Ser AlaGly Thr Asn Leu Glu Gly Lys Ala 420 425 430 gtc gaa att aca aca ctt aaagaa ctt aaa aac gct tta aaa taa 1341 Val Glu Ile Thr Thr Leu Lys Glu LeuLys Asn Ala Leu Lys * 435 440 445 64 446 PRT Artificial SequenceChimeric protein 64 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr SerAla Asn Ser 1 5 10 15 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr GluIle Ser Lys Lys 20 25 30 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val LysGlu Val Glu Ala 35 40 45 Leu Leu Ser Ser Ile Asp Glu Leu Ala Lys Ala IleGly Lys Lys Ile 50 55 60 Lys Asn Asp Gly Ser Leu Asp Asn Glu Ala Asn ArgAsn Glu Ser Leu 65 70 75 80 Leu Ala Gly Ala Tyr Thr Ile Ser Thr Leu IleThr Gln Lys Leu Ser 85 90 95 Lys Leu Asn Gly Ser Glu Gly Leu Lys Glu LysIle Ala Ala Ala Lys 100 105 110 Lys Cys Ser Glu Glu Phe Ser Thr Lys LeuLys Asp Asn His Ala Gln 115 120 125 Leu Gly Ile Gln Gly Val Thr Asp GluAsn Ala Lys Lys Ala Ile Leu 130 135 140 Lys Ala Asn Ala Ala Gly Lys AspLys Gly Val Glu Glu Leu Glu Lys 145 150 155 160 Leu Ser Gly Ser Leu GluSer Leu Ser Lys Ala Ala Lys Glu Met Leu 165 170 175 Ala Asn Ser Val LysGlu Leu Thr Ser Pro Val Val His Gly Lys Gln 180 185 190 Asn Val Ser SerLeu Asp Glu Lys Asn Ser Val Ser Val Asp Leu Pro 195 200 205 Gly Glu MetLys Val Leu Val Ser Lys Glu Lys Asn Lys Asp Gly Lys 210 215 220 Tyr AspLeu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys Gly Thr Ser 225 230 235 240Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys Ala Asp Lys 245 250255 Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln Thr Thr Leu 260265 270 Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys Lys Val Thr275 280 285 Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu Lys GlyGlu 290 295 300 Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg LeuGlu Tyr 305 310 315 320 Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala LysGlu Val Leu Lys 325 330 335 Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala GluLys Thr Thr Leu Val 340 345 350 Val Lys Glu Gly Thr Val Thr Leu Ser LysAsn Ile Ser Lys Ser Gly 355 360 365 Glu Val Ser Val Glu Leu Asn Asp ThrAsp Ser Ser Ala Ala Thr Lys 370 375 380 Lys Thr Ala Ala Trp Asn Ser LysThr Ser Thr Leu Thr Ile Ser Val 385 390 395 400 Asn Ser Gln Lys Thr LysAsn Leu Val Phe Thr Lys Glu Asp Thr Ile 405 410 415 Thr Val Gln Lys TyrAsp Ser Ala Gly Thr Asn Leu Glu Gly Lys Ala 420 425 430 Val Glu Ile ThrThr Leu Lys Glu Leu Lys Asn Ala Leu Lys 435 440 445 65 1362 DNAArtificial Sequence Chimeric nucleic acid 65 atg gct tgt aat aat tca gggaaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly LysAsp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tct gtt aaa gggcct aat ctt aca gaa ata aat aaa aaa 96 Ala Asp Glu Ser Val Lys Gly ProAsn Leu Thr Glu Ile Asn Lys Lys 20 25 30 att acg tca tct aat gcg gtt ttactt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Ser Ser Asn Ala Val Leu LeuAla Val Lys Glu Val Glu Ala 35 40 45 ttg ctg gat tct ata gat gaa att gctgct aaa gct att ggt aaa aaa 192 Leu Leu Asp Ser Ile Asp Glu Ile Ala AlaLys Ala Ile Gly Lys Lys 50 55 60 ata cac caa aat aat ggt ttg gat acc gaaaat aat cac aat gga tca 240 Ile His Gln Asn Asn Gly Leu Asp Thr Glu AsnAsn His Asn Gly Ser 65 70 75 80 ttg tta gcg gga gct tat gca ata tca acccta ata aaa caa aaa tta 288 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr LeuIle Lys Gln Lys Leu 85 90 95 gat gga ttg aaa aat gaa gga tta aag gaa aaaatt gat gcg gct aag 336 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys IleAsp Ala Ala Lys 100 105 110 aaa tgt tct gaa aca ttt act aat aaa tta aaagaa aaa cac aca gat 384 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys GluLys His Thr Asp 115 120 125 ctt ggt aaa gaa ggt gtt act gat gct gat gcaaaa gaa gcc att tta 432 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala LysGlu Ala Ile Leu 130 135 140 aaa gca aat ggt act aaa act aaa ggt gct gaagaa ctt gga aaa tta 480 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu GluLeu Gly Lys Leu 145 150 155 160 ttt gaa tca gta gag gtc ttg tca aaa gcagct aaa gag atg ctt gct 528 Phe Glu Ser Val Glu Val Leu Ser Lys Ala AlaLys Glu Met Leu Ala 165 170 175 aat tca gtt aaa gag ctt aca agc cct gttgtg gca gaa agt cca aaa 576 Asn Ser Val Lys Glu Leu Thr Ser Pro Val ValAla Glu Ser Pro Lys 180 185 190 aaa cct tcc atg gcc aag caa aat gtt agcagc ctt gac gag aaa aac 624 Lys Pro Ser Met Ala Lys Gln Asn Val Ser SerLeu Asp Glu Lys Asn 195 200 205 agc gtt tca gta gat ttg cct ggt gaa atgaaa gtt ctt gta agc aaa 672 Ser Val Ser Val Asp Leu Pro Gly Glu Met LysVal Leu Val Ser Lys 210 215 220 gaa aaa aac aaa gac ggc aag tac gat ctaatt gca aca gta gac aag 720 Glu Lys Asn Lys Asp Gly Lys Tyr Asp Leu IleAla Thr Val Asp Lys 225 230 235 240 ctt gag ctt aaa gga act tct gat aaaaac aat gga tct gga gta ctt 768 Leu Glu Leu Lys Gly Thr Ser Asp Lys AsnAsn Gly Ser Gly Val Leu 245 250 255 gaa ggc gta aaa gct gac aaa agt aaagta aaa tta aca att tct gac 816 Glu Gly Val Lys Ala Asp Lys Ser Lys ValLys Leu Thr Ile Ser Asp 260 265 270 gat cta ggt caa acc aca ctt gaa gttttc aaa gaa gat ggc aaa aca 864 Asp Leu Gly Gln Thr Thr Leu Glu Val PheLys Glu Asp Gly Lys Thr 275 280 285 cta gta tca aaa aaa gta act tcc aaagac aag tca tca aca gaa gaa 912 Leu Val Ser Lys Lys Val Thr Ser Lys AspLys Ser Ser Thr Glu Glu 290 295 300 aaa ttc aat gaa aaa ggt gaa gta tctgaa aaa ata ata aca aga gca 960 Lys Phe Asn Glu Lys Gly Glu Val Ser GluLys Ile Ile Thr Arg Ala 305 310 315 320 gac gga acc aga ctt gaa tac acagga att aaa agc gat gga tct gga 1008 Asp Gly Thr Arg Leu Glu Tyr Thr GlyIle Lys Ser Asp Gly Ser Gly 325 330 335 aaa gct aaa gag gtt tta aaa ggctat gtt ctt gaa gga act cta act 1056 Lys Ala Lys Glu Val Leu Lys Gly TyrVal Leu Glu Gly Thr Leu Thr 340 345 350 gct gaa aaa aca aca ttg gtg gttaaa gaa gga act gtt act tta agc 1104 Ala Glu Lys Thr Thr Leu Val Val LysGlu Gly Thr Val Thr Leu Ser 355 360 365 aaa aat att tca aaa tct ggg gaagtt tca gtt gaa ctt aat gac act 1152 Lys Asn Ile Ser Lys Ser Gly Glu ValSer Val Glu Leu Asn Asp Thr 370 375 380 gac agt agt gct gct act aaa aaaact gca gct tgg aat tca aaa act 1200 Asp Ser Ser Ala Ala Thr Lys Lys ThrAla Ala Trp Asn Ser Lys Thr 385 390 395 400 tct act tta aca att agt gttaac agc aaa aaa act aca caa ctt gtg 1248 Ser Thr Leu Thr Ile Ser Val AsnSer Lys Lys Thr Thr Gln Leu Val 405 410 415 ttt act aaa caa gac aca ataact gta caa aaa tac gac tcc gca ggt 1296 Phe Thr Lys Gln Asp Thr Ile ThrVal Gln Lys Tyr Asp Ser Ala Gly 420 425 430 acc aat tta gaa ggc aca gcagtc gaa att aaa aca ctt gat gaa ctt 1344 Thr Asn Leu Glu Gly Thr Ala ValGlu Ile Lys Thr Leu Asp Glu Leu 435 440 445 aaa aac gct tta aaa taa 1362Lys Asn Ala Leu Lys * 450 66 453 PRT Artificial Sequence Chimericprotein 66 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala AsnSer 1 5 10 15 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile AsnLys Lys 20 25 30 Ile Thr Ser Ser Asn Ala Val Leu Leu Ala Val Lys Glu ValGlu Ala 35 40 45 Leu Leu Asp Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile GlyLys Lys 50 55 60 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Asn Asn His AsnGly Ser 65 70 75 80 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile LysGln Lys Leu 85 90 95 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile AspAla Ala Lys 100 105 110 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys GluLys His Thr Asp 115 120 125 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp AlaLys Glu Ala Ile Leu 130 135 140 Lys Ala Asn Gly Thr Lys Thr Lys Gly AlaGlu Glu Leu Gly Lys Leu 145 150 155 160 Phe Glu Ser Val Glu Val Leu SerLys Ala Ala Lys Glu Met Leu Ala 165 170 175 Asn Ser Val Lys Glu Leu ThrSer Pro Val Val Ala Glu Ser Pro Lys 180 185 190 Lys Pro Ser Met Ala LysGln Asn Val Ser Ser Leu Asp Glu Lys Asn 195 200 205 Ser Val Ser Val AspLeu Pro Gly Glu Met Lys Val Leu Val Ser Lys 210 215 220 Glu Lys Asn LysAsp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys 225 230 235 240 Leu GluLeu Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu 245 250 255 GluGly Val Lys Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp 260 265 270Asp Leu Gly Gln Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr 275 280285 Leu Val Ser Lys Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu 290295 300 Lys Phe Asn Glu Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala305 310 315 320 Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp GlySer Gly 325 330 335 Lys Ala Lys Glu Val Leu Lys Gly Tyr Val Leu Glu GlyThr Leu Thr 340 345 350 Ala Glu Lys Thr Thr Leu Val Val Lys Glu Gly ThrVal Thr Leu Ser 355 360 365 Lys Asn Ile Ser Lys Ser Gly Glu Val Ser ValGlu Leu Asn Asp Thr 370 375 380 Asp Ser Ser Ala Ala Thr Lys Lys Thr AlaAla Trp Asn Ser Lys Thr 385 390 395 400 Ser Thr Leu Thr Ile Ser Val AsnSer Lys Lys Thr Thr Gln Leu Val 405 410 415 Phe Thr Lys Gln Asp Thr IleThr Val Gln Lys Tyr Asp Ser Ala Gly 420 425 430 Thr Asn Leu Glu Gly ThrAla Val Glu Ile Lys Thr Leu Asp Glu Leu 435 440 445 Lys Asn Ala Leu Lys450 67 1341 DNA Artificial Sequence Chimeric nucleic acid 67 atg gct tgtaat aat tca ggg aaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys AsnAsn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gagtct gtt aaa ggg cct aat ctt aca gaa ata agt aaa aaa 96 Ala Asp Glu SerVal Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 att acg gat tctaat gcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Asp Ser AsnAla Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttg ctg tca tct atagat gag ctt gct aaa gct att ggt aaa aaa ata 192 Leu Leu Ser Ser Ile AspGlu Leu Ala Lys Ala Ile Gly Lys Lys Ile 50 55 60 aaa aac gat ggt agt ttagat aat gaa gca aat cgc aac gag tca ttg 240 Lys Asn Asp Gly Ser Leu AspAsn Glu Ala Asn Arg Asn Glu Ser Leu 65 70 75 80 tta gca gga gct tat acaata tca acc tta ata aca caa aaa tta agt 288 Leu Ala Gly Ala Tyr Thr IleSer Thr Leu Ile Thr Gln Lys Leu Ser 85 90 95 aaa tta aac gga tca gaa ggttta aag gaa aag att gcc gca gct aag 336 Lys Leu Asn Gly Ser Glu Gly LeuLys Glu Lys Ile Ala Ala Ala Lys 100 105 110 aaa tgc tct gaa gag ttt agtact aaa cta aaa gat aat cat gca cag 384 Lys Cys Ser Glu Glu Phe Ser ThrLys Leu Lys Asp Asn His Ala Gln 115 120 125 ctt ggt ata cag ggc gtt actgat gaa aat gca aaa aaa gct att tta 432 Leu Gly Ile Gln Gly Val Thr AspGlu Asn Ala Lys Lys Ala Ile Leu 130 135 140 aaa gca aat gca gcg ggt aaagat aag ggc gtt gaa gaa ctt gaa aag 480 Lys Ala Asn Ala Ala Gly Lys AspLys Gly Val Glu Glu Leu Glu Lys 145 150 155 160 ttg tcc gga tca tta gaaagc tta tca aaa gca gct aaa gag atg ctt 528 Leu Ser Gly Ser Leu Glu SerLeu Ser Lys Ala Ala Lys Glu Met Leu 165 170 175 gct aat tca gtt aaa gagctt aca agc cct gtt gtc cat ggc aag caa 576 Ala Asn Ser Val Lys Glu LeuThr Ser Pro Val Val His Gly Lys Gln 180 185 190 aat gtt agc agc ctt gacgag aaa aac agc gtt tca gta gat ttg cct 624 Asn Val Ser Ser Leu Asp GluLys Asn Ser Val Ser Val Asp Leu Pro 195 200 205 ggt gaa atg aaa gtt cttgta agc aaa gaa aaa aac aaa gac ggc aag 672 Gly Glu Met Lys Val Leu ValSer Lys Glu Lys Asn Lys Asp Gly Lys 210 215 220 tac gat cta att gca acagta gac aag ctt gag ctt aaa gga act tct 720 Tyr Asp Leu Ile Ala Thr ValAsp Lys Leu Glu Leu Lys Gly Thr Ser 225 230 235 240 gat aaa aac aat ggatct gga gta ctt gaa ggc gta aaa gct gac aaa 768 Asp Lys Asn Asn Gly SerGly Val Leu Glu Gly Val Lys Ala Asp Lys 245 250 255 agt aaa gta aaa ttaaca att tct gac gat cta ggt caa acc aca ctt 816 Ser Lys Val Lys Leu ThrIle Ser Asp Asp Leu Gly Gln Thr Thr Leu 260 265 270 gaa gtt ttc aaa gaagat ggc aaa aca cta gta tca aaa aaa gta act 864 Glu Val Phe Lys Glu AspGly Lys Thr Leu Val Ser Lys Lys Val Thr 275 280 285 tcc aaa gac aag tcatca aca gaa gaa aaa ttc aat gaa aaa ggt gaa 912 Ser Lys Asp Lys Ser SerThr Glu Glu Lys Phe Asn Glu Lys Gly Glu 290 295 300 gta tct gaa aaa ataata aca aga gca gac gga acc aga ctt gaa tac 960 Val Ser Glu Lys Ile IleThr Arg Ala Asp Gly Thr Arg Leu Glu Tyr 305 310 315 320 aca gga att aaaagc gat gga tct gga aaa gct aaa gag gtt tta aaa 1008 Thr Gly Ile Lys SerAsp Gly Ser Gly Lys Ala Lys Glu Val Leu Lys 325 330 335 ggc tat gtt cttgaa gga act cta act gct gaa aaa aca aca ttg gtg 1056 Gly Tyr Val Leu GluGly Thr Leu Thr Ala Glu Lys Thr Thr Leu Val 340 345 350 gtt aaa gaa ggaact gtt act tta agc aaa aat att tca aaa tct ggg 1104 Val Lys Glu Gly ThrVal Thr Leu Ser Lys Asn Ile Ser Lys Ser Gly 355 360 365 gaa gtt tca gttgaa ctt aat gac act gac agt agt gct gct act aaa 1152 Glu Val Ser Val GluLeu Asn Asp Thr Asp Ser Ser Ala Ala Thr Lys 370 375 380 aaa act gca gcttgg aat tca aaa act tct act tta aca att agt gtt 1200 Lys Thr Ala Ala TrpAsn Ser Lys Thr Ser Thr Leu Thr Ile Ser Val 385 390 395 400 aac agc aaaaaa act aca caa ctt gtg ttt act aaa caa tac aca ata 1248 Asn Ser Lys LysThr Thr Gln Leu Val Phe Thr Lys Gln Tyr Thr Ile 405 410 415 act gta aaacaa tac gac tcc gca ggt acc aat tta gaa ggc aca gca 1296 Thr Val Lys GlnTyr Asp Ser Ala Gly Thr Asn Leu Glu Gly Thr Ala 420 425 430 gtc gaa attaaa aca ctt gat gaa ctt aaa aac gct tta aaa taa 1341 Val Glu Ile Lys ThrLeu Asp Glu Leu Lys Asn Ala Leu Lys * 435 440 445 68 446 PRT ArtificialSequence Chimeric protein 68 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly AsnThr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser Val Lys Gly Pro Asn LeuThr Glu Ile Ser Lys Lys 20 25 30 Ile Thr Asp Ser Asn Ala Val Leu Leu AlaVal Lys Glu Val Glu Ala 35 40 45 Leu Leu Ser Ser Ile Asp Glu Leu Ala LysAla Ile Gly Lys Lys Ile 50 55 60 Lys Asn Asp Gly Ser Leu Asp Asn Glu AlaAsn Arg Asn Glu Ser Leu 65 70 75 80 Leu Ala Gly Ala Tyr Thr Ile Ser ThrLeu Ile Thr Gln Lys Leu Ser 85 90 95 Lys Leu Asn Gly Ser Glu Gly Leu LysGlu Lys Ile Ala Ala Ala Lys 100 105 110 Lys Cys Ser Glu Glu Phe Ser ThrLys Leu Lys Asp Asn His Ala Gln 115 120 125 Leu Gly Ile Gln Gly Val ThrAsp Glu Asn Ala Lys Lys Ala Ile Leu 130 135 140 Lys Ala Asn Ala Ala GlyLys Asp Lys Gly Val Glu Glu Leu Glu Lys 145 150 155 160 Leu Ser Gly SerLeu Glu Ser Leu Ser Lys Ala Ala Lys Glu Met Leu 165 170 175 Ala Asn SerVal Lys Glu Leu Thr Ser Pro Val Val His Gly Lys Gln 180 185 190 Asn ValSer Ser Leu Asp Glu Lys Asn Ser Val Ser Val Asp Leu Pro 195 200 205 GlyGlu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys Asp Gly Lys 210 215 220Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys Gly Thr Ser 225 230235 240 Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys Ala Asp Lys245 250 255 Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln Thr ThrLeu 260 265 270 Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys LysVal Thr 275 280 285 Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn GluLys Gly Glu 290 295 300 Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly ThrArg Leu Glu Tyr 305 310 315 320 Thr Gly Ile Lys Ser Asp Gly Ser Gly LysAla Lys Glu Val Leu Lys 325 330 335 Gly Tyr Val Leu Glu Gly Thr Leu ThrAla Glu Lys Thr Thr Leu Val 340 345 350 Val Lys Glu Gly Thr Val Thr LeuSer Lys Asn Ile Ser Lys Ser Gly 355 360 365 Glu Val Ser Val Glu Leu AsnAsp Thr Asp Ser Ser Ala Ala Thr Lys 370 375 380 Lys Thr Ala Ala Trp AsnSer Lys Thr Ser Thr Leu Thr Ile Ser Val 385 390 395 400 Asn Ser Lys LysThr Thr Gln Leu Val Phe Thr Lys Gln Tyr Thr Ile 405 410 415 Thr Val LysGln Tyr Asp Ser Ala Gly Thr Asn Leu Glu Gly Thr Ala 420 425 430 Val GluIle Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu Lys 435 440 445 69 1365 DNAArtificial Sequence Chimeric nucleic acid 69 atg gct tgt aat aat tca gggaaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys Asn Asn Ser Gly LysAsp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tct gtt aaa gggcct aat ctt aca gaa ata agt aaa aaa 96 Ala Asp Glu Ser Val Lys Gly ProAsn Leu Thr Glu Ile Ser Lys Lys 20 25 30 att acg gat tct aat gcg gtt ttactt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Asp Ser Asn Ala Val Leu LeuAla Val Lys Glu Val Glu Ala 35 40 45 ttg ctg tca tct ata gat gaa att gctgct aaa gct att ggt aaa aaa 192 Leu Leu Ser Ser Ile Asp Glu Ile Ala AlaLys Ala Ile Gly Lys Lys 50 55 60 ata cac caa aat aat ggt ttg gat acc gaatat aat cac aat gga tca 240 Ile His Gln Asn Asn Gly Leu Asp Thr Glu TyrAsn His Asn Gly Ser 65 70 75 80 ttg tta gcg gga gct tat gca ata tca acccta ata aaa caa aaa tta 288 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr LeuIle Lys Gln Lys Leu 85 90 95 gat gga ttg aaa aat gaa gga tta aag gaa aaaatt gat gcg gct aag 336 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys IleAsp Ala Ala Lys 100 105 110 aaa tgt tct gaa aca ttt act aat aaa tta aaagaa aaa cac aca gat 384 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys GluLys His Thr Asp 115 120 125 ctt ggt aaa gaa ggt gtt act gat gct gat gcaaaa gaa gcc att tta 432 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala LysGlu Ala Ile Leu 130 135 140 aaa aca aat ggt act aaa act aaa ggt gct gaagaa ctt gga aaa tta 480 Lys Thr Asn Gly Thr Lys Thr Lys Gly Ala Glu GluLeu Gly Lys Leu 145 150 155 160 ttt gaa tca gta gag gtc ttg tca aaa gcagct aaa gag atg ctt gct 528 Phe Glu Ser Val Glu Val Leu Ser Lys Ala AlaLys Glu Met Leu Ala 165 170 175 aat tca gtt aaa gag ctt aca agc cct gttgtg gca gaa agt cca aaa 576 Asn Ser Val Lys Glu Leu Thr Ser Pro Val ValAla Glu Ser Pro Lys 180 185 190 aaa cct tcc atg gcc aag caa aat gtt agcagc ctt gat gaa aaa aat 624 Lys Pro Ser Met Ala Lys Gln Asn Val Ser SerLeu Asp Glu Lys Asn 195 200 205 agc gtt tca gta gat tta cct ggt gga atgaca gtt ctt gta agt aaa 672 Ser Val Ser Val Asp Leu Pro Gly Gly Met ThrVal Leu Val Ser Lys 210 215 220 gaa aaa gac aaa gac ggt aaa tac agt ctagag gca aca gta gac aag 720 Glu Lys Asp Lys Asp Gly Lys Tyr Ser Leu GluAla Thr Val Asp Lys 225 230 235 240 ctt gag ctt aaa gga act tct gat aaaaac aac ggt tct gga aca ctt 768 Leu Glu Leu Lys Gly Thr Ser Asp Lys AsnAsn Gly Ser Gly Thr Leu 245 250 255 gaa ggt gaa aaa act gac aaa agt aaagta aaa tta aca att gct gat 816 Glu Gly Glu Lys Thr Asp Lys Ser Lys ValLys Leu Thr Ile Ala Asp 260 265 270 gac cta agt caa act aaa ttt gaa attttc aaa gaa gat gcc aaa aca 864 Asp Leu Ser Gln Thr Lys Phe Glu Ile PheLys Glu Asp Ala Lys Thr 275 280 285 tta gta tca aaa aaa gta acc ctt aaagac aag tca tca aca gaa gaa 912 Leu Val Ser Lys Lys Val Thr Leu Lys AspLys Ser Ser Thr Glu Glu 290 295 300 aaa ttc aac gaa aag ggt gaa aca tctgaa aaa aca ata gta aga gca 960 Lys Phe Asn Glu Lys Gly Glu Thr Ser GluLys Thr Ile Val Arg Ala 305 310 315 320 aat gga acc aga ctt gaa tac acagac ata aaa agc gat gga tcc gga 1008 Asn Gly Thr Arg Leu Glu Tyr Thr AspIle Lys Ser Asp Gly Ser Gly 325 330 335 aaa gct aaa gaa gtt tta aaa gacttt act ctt gaa gga act cta gct 1056 Lys Ala Lys Glu Val Leu Lys Asp PheThr Leu Glu Gly Thr Leu Ala 340 345 350 gct gac ggc aaa aca aca ttg aaagtt aca gaa ggc act gtt gtt tta 1104 Ala Asp Gly Lys Thr Thr Leu Lys ValThr Glu Gly Thr Val Val Leu 355 360 365 agc aag aac att tta aaa tcc ggagaa ata aca gtt gca ctt gat gac 1152 Ser Lys Asn Ile Leu Lys Ser Gly GluIle Thr Val Ala Leu Asp Asp 370 375 380 tct gac act act cag gct act aaaaaa act gga aaa tgg gat tca aat 1200 Ser Asp Thr Thr Gln Ala Thr Lys LysThr Gly Lys Trp Asp Ser Asn 385 390 395 400 act tcc act tta aca att agtgtg aat agc aaa aaa act aaa aac att 1248 Thr Ser Thr Leu Thr Ile Ser ValAsn Ser Lys Lys Thr Lys Asn Ile 405 410 415 gta ttt aca aaa gaa gac acaata aca gta caa aaa tac gac tca gca 1296 Val Phe Thr Lys Glu Asp Thr IleThr Val Gln Lys Tyr Asp Ser Ala 420 425 430 ggc acc aat cta gaa ggc aacgca gtc gaa att aaa aca ctt gat gaa 1344 Gly Thr Asn Leu Glu Gly Asn AlaVal Glu Ile Lys Thr Leu Asp Glu 435 440 445 ctt aaa aac gct tta aaa tag1365 Leu Lys Asn Ala Leu Lys * 450 70 454 PRT Artificial SequenceChimeric protein 70 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr SerAla Asn Ser 1 5 10 15 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr GluIle Ser Lys Lys 20 25 30 Ile Thr Asp Ser Asn Ala Val Leu Leu Ala Val LysGlu Val Glu Ala 35 40 45 Leu Leu Ser Ser Ile Asp Glu Ile Ala Ala Lys AlaIle Gly Lys Lys 50 55 60 Ile His Gln Asn Asn Gly Leu Asp Thr Glu Tyr AsnHis Asn Gly Ser 65 70 75 80 Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr LeuIle Lys Gln Lys Leu 85 90 95 Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu LysIle Asp Ala Ala Lys 100 105 110 Lys Cys Ser Glu Thr Phe Thr Asn Lys LeuLys Glu Lys His Thr Asp 115 120 125 Leu Gly Lys Glu Gly Val Thr Asp AlaAsp Ala Lys Glu Ala Ile Leu 130 135 140 Lys Thr Asn Gly Thr Lys Thr LysGly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 Phe Glu Ser Val Glu ValLeu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 Asn Ser Val Lys GluLeu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 Lys Pro Ser MetAla Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn 195 200 205 Ser Val SerVal Asp Leu Pro Gly Gly Met Thr Val Leu Val Ser Lys 210 215 220 Glu LysAsp Lys Asp Gly Lys Tyr Ser Leu Glu Ala Thr Val Asp Lys 225 230 235 240Leu Glu Leu Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Thr Leu 245 250255 Glu Gly Glu Lys Thr Asp Lys Ser Lys Val Lys Leu Thr Ile Ala Asp 260265 270 Asp Leu Ser Gln Thr Lys Phe Glu Ile Phe Lys Glu Asp Ala Lys Thr275 280 285 Leu Val Ser Lys Lys Val Thr Leu Lys Asp Lys Ser Ser Thr GluGlu 290 295 300 Lys Phe Asn Glu Lys Gly Glu Thr Ser Glu Lys Thr Ile ValArg Ala 305 310 315 320 Asn Gly Thr Arg Leu Glu Tyr Thr Asp Ile Lys SerAsp Gly Ser Gly 325 330 335 Lys Ala Lys Glu Val Leu Lys Asp Phe Thr LeuGlu Gly Thr Leu Ala 340 345 350 Ala Asp Gly Lys Thr Thr Leu Lys Val ThrGlu Gly Thr Val Val Leu 355 360 365 Ser Lys Asn Ile Leu Lys Ser Gly GluIle Thr Val Ala Leu Asp Asp 370 375 380 Ser Asp Thr Thr Gln Ala Thr LysLys Thr Gly Lys Trp Asp Ser Asn 385 390 395 400 Thr Ser Thr Leu Thr IleSer Val Asn Ser Lys Lys Thr Lys Asn Ile 405 410 415 Val Phe Thr Lys GluAsp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala 420 425 430 Gly Thr Asn LeuGlu Gly Asn Ala Val Glu Ile Lys Thr Leu Asp Glu 435 440 445 Leu Lys AsnAla Leu Lys 450 71 1344 DNA Artificial Sequence Chimeric nucleic acid 71atg gct tgt aat aat tca ggg aaa gat ggg aat aca tct gca aat tct 48 MetAla Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15gct gat gag tct gtt aaa ggg cct aat ctt aca gaa ata agt aaa aaa 96 AlaAsp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 attacg gat tct aat gcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 Ile ThrAsp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttg ctgtca tct ata gat gag ctt gct aaa gct att ggt aaa aaa ata 192 Leu Leu SerSer Ile Asp Glu Leu Ala Lys Ala Ile Gly Lys Lys Ile 50 55 60 aaa aac gatggt agt tta gat aat gaa gca aat cgc aac gag tca ttg 240 Lys Asn Asp GlySer Leu Asp Asn Glu Ala Asn Arg Asn Glu Ser Leu 65 70 75 80 tta gca ggagct tat aca ata tca acc tta ata aca caa aaa tta agt 288 Leu Ala Gly AlaTyr Thr Ile Ser Thr Leu Ile Thr Gln Lys Leu Ser 85 90 95 aaa tta aac ggatca gaa ggt tta aag gaa aag att gcc gca gct aag 336 Lys Leu Asn Gly SerGlu Gly Leu Lys Glu Lys Ile Ala Ala Ala Lys 100 105 110 aaa tgc tct gaagag ttt agt act aaa cta aaa gat aat cat gca cag 384 Lys Cys Ser Glu GluPhe Ser Thr Lys Leu Lys Asp Asn His Ala Gln 115 120 125 ctt ggt ata cagggc gtt act gat gaa aat gca aaa aaa gct att tta 432 Leu Gly Ile Gln GlyVal Thr Asp Glu Asn Ala Lys Lys Ala Ile Leu 130 135 140 aaa gca aat gcagcg ggt aaa gat aag ggc gtt gaa gaa ctt gaa aag 480 Lys Ala Asn Ala AlaGly Lys Asp Lys Gly Val Glu Glu Leu Glu Lys 145 150 155 160 ttg tcc ggatca tta gaa agc tta tca aaa gca gct aaa gag atg ctt 528 Leu Ser Gly SerLeu Glu Ser Leu Ser Lys Ala Ala Lys Glu Met Leu 165 170 175 gct aat tcagtt aaa gag ctt aca agc cct gtt gtc cat ggc aag caa 576 Ala Asn Ser ValLys Glu Leu Thr Ser Pro Val Val His Gly Lys Gln 180 185 190 aat gtt agcagc ctt gat gaa aaa aat agc gtt tca gta gat tta cct 624 Asn Val Ser SerLeu Asp Glu Lys Asn Ser Val Ser Val Asp Leu Pro 195 200 205 ggt gga atgaca gtt ctt gta agt aaa gaa aaa gac aaa gac ggt aaa 672 Gly Gly Met ThrVal Leu Val Ser Lys Glu Lys Asp Lys Asp Gly Lys 210 215 220 tac agt ctagag gca aca gta gac aag ctt gag ctt aaa gga act tct 720 Tyr Ser Leu GluAla Thr Val Asp Lys Leu Glu Leu Lys Gly Thr Ser 225 230 235 240 gat aaaaac aac ggt tct gga aca ctt gaa ggt gaa aaa act gac aaa 768 Asp Lys AsnAsn Gly Ser Gly Thr Leu Glu Gly Glu Lys Thr Asp Lys 245 250 255 agt aaagta aaa tta aca att gct gat gac cta agt caa act aaa ttt 816 Ser Lys ValLys Leu Thr Ile Ala Asp Asp Leu Ser Gln Thr Lys Phe 260 265 270 gaa attttc aaa gaa gat gcc aaa aca tta gta tca aaa aaa gta acc 864 Glu Ile PheLys Glu Asp Ala Lys Thr Leu Val Ser Lys Lys Val Thr 275 280 285 ctt aaagac aag tca tca aca gaa gaa aaa ttc aac gaa aag ggt gaa 912 Leu Lys AspLys Ser Ser Thr Glu Glu Lys Phe Asn Glu Lys Gly Glu 290 295 300 aca tctgaa aaa aca ata gta aga gca aat gga acc aga ctt gaa tac 960 Thr Ser GluLys Thr Ile Val Arg Ala Asn Gly Thr Arg Leu Glu Tyr 305 310 315 320 acagac ata aaa agc gat gga tcc gga aaa gct aaa gaa gtt tta aaa 1008 Thr AspIle Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu Val Leu Lys 325 330 335 gacttt act ctt gaa gga act cta gct gct gac ggc aaa aca aca ttg 1056 Asp PheThr Leu Glu Gly Thr Leu Ala Ala Asp Gly Lys Thr Thr Leu 340 345 350 aaagtt aca gaa ggc act gtt gtt tta agc aag aac att tta aaa tcc 1104 Lys ValThr Glu Gly Thr Val Val Leu Ser Lys Asn Ile Leu Lys Ser 355 360 365 ggagaa ata aca gtt gca ctt gat gac tct gac act act cag gct act 1152 Gly GluIle Thr Val Ala Leu Asp Asp Ser Asp Thr Thr Gln Ala Thr 370 375 380 aaaaaa act gga aaa tgg gat tca aat act tcc act tta aca att agt 1200 Lys LysThr Gly Lys Trp Asp Ser Asn Thr Ser Thr Leu Thr Ile Ser 385 390 395 400gtg aat agc aaa aaa act aaa aac att gta ttt aca aaa gaa gac aca 1248 ValAsn Ser Lys Lys Thr Lys Asn Ile Val Phe Thr Lys Glu Asp Thr 405 410 415ata aca gta caa aaa tac gac tca gca ggc acc aat cta gaa ggc aac 1296 IleThr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu Gly Asn 420 425 430gca gtc gaa att aaa aca ctt gat gaa ctt aaa aac gct tta aaa tag 1344 AlaVal Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu Lys * 435 440 445 72447 PRT Artificial Sequence Chimeric protein 72 Met Ala Cys Asn Asn SerGly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser ValLys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 Ile Thr Asp Ser AsnAla Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu Ser Ser IleAsp Glu Leu Ala Lys Ala Ile Gly Lys Lys Ile 50 55 60 Lys Asn Asp Gly SerLeu Asp Asn Glu Ala Asn Arg Asn Glu Ser Leu 65 70 75 80 Leu Ala Gly AlaTyr Thr Ile Ser Thr Leu Ile Thr Gln Lys Leu Ser 85 90 95 Lys Leu Asn GlySer Glu Gly Leu Lys Glu Lys Ile Ala Ala Ala Lys 100 105 110 Lys Cys SerGlu Glu Phe Ser Thr Lys Leu Lys Asp Asn His Ala Gln 115 120 125 Leu GlyIle Gln Gly Val Thr Asp Glu Asn Ala Lys Lys Ala Ile Leu 130 135 140 LysAla Asn Ala Ala Gly Lys Asp Lys Gly Val Glu Glu Leu Glu Lys 145 150 155160 Leu Ser Gly Ser Leu Glu Ser Leu Ser Lys Ala Ala Lys Glu Met Leu 165170 175 Ala Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val His Gly Lys Gln180 185 190 Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val Asp LeuPro 195 200 205 Gly Gly Met Thr Val Leu Val Ser Lys Glu Lys Asp Lys AspGly Lys 210 215 220 Tyr Ser Leu Glu Ala Thr Val Asp Lys Leu Glu Leu LysGly Thr Ser 225 230 235 240 Asp Lys Asn Asn Gly Ser Gly Thr Leu Glu GlyGlu Lys Thr Asp Lys 245 250 255 Ser Lys Val Lys Leu Thr Ile Ala Asp AspLeu Ser Gln Thr Lys Phe 260 265 270 Glu Ile Phe Lys Glu Asp Ala Lys ThrLeu Val Ser Lys Lys Val Thr 275 280 285 Leu Lys Asp Lys Ser Ser Thr GluGlu Lys Phe Asn Glu Lys Gly Glu 290 295 300 Thr Ser Glu Lys Thr Ile ValArg Ala Asn Gly Thr Arg Leu Glu Tyr 305 310 315 320 Thr Asp Ile Lys SerAsp Gly Ser Gly Lys Ala Lys Glu Val Leu Lys 325 330 335 Asp Phe Thr LeuGlu Gly Thr Leu Ala Ala Asp Gly Lys Thr Thr Leu 340 345 350 Lys Val ThrGlu Gly Thr Val Val Leu Ser Lys Asn Ile Leu Lys Ser 355 360 365 Gly GluIle Thr Val Ala Leu Asp Asp Ser Asp Thr Thr Gln Ala Thr 370 375 380 LysLys Thr Gly Lys Trp Asp Ser Asn Thr Ser Thr Leu Thr Ile Ser 385 390 395400 Val Asn Ser Lys Lys Thr Lys Asn Ile Val Phe Thr Lys Glu Asp Thr 405410 415 Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu Gly Asn420 425 430 Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu Lys435 440 445 73 1305 DNA Artificial Sequence Chimeric nucleic acid 73 atggct tgt aat aat tca gga aaa gat ggg aat aca tct gca aat tct 48 Met AlaCys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gctgat gag tct gtt aaa ggg cct aat ctt aca gaa ata agt aaa aaa 96 Ala AspGlu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 att acagaa tct aac gca gtt gtt ctg gct gtg aaa gaa att gaa act 144 Ile Thr GluSer Asn Ala Val Val Leu Ala Val Lys Glu Ile Glu Thr 35 40 45 ttg ctt gcatct ata gat gaa ctt gct act aaa gct att ggt aaa aaa 192 Leu Leu Ala SerIle Asp Glu Leu Ala Thr Lys Ala Ile Gly Lys Lys 50 55 60 ata caa caa aatggt ggt tta gct gtc gaa gcg ggg cat aat gga aca 240 Ile Gln Gln Asn GlyGly Leu Ala Val Glu Ala Gly His Asn Gly Thr 65 70 75 80 ttg tta gca ggtgct tat aca ata tca aaa cta ata aca caa aaa tta 288 Leu Leu Ala Gly AlaTyr Thr Ile Ser Lys Leu Ile Thr Gln Lys Leu 85 90 95 gat gga ttg aaa aattca gaa aaa tta aag gaa aaa att gaa aat gct 336 Asp Gly Leu Lys Asn SerGlu Lys Leu Lys Glu Lys Ile Glu Asn Ala 100 105 110 aag aaa tgt tct gaagat ttt act aaa aaa cta gaa gga gaa cat gcg 384 Lys Lys Cys Ser Glu AspPhe Thr Lys Lys Leu Glu Gly Glu His Ala 115 120 125 caa ctt gga att gaaaat gtt act gat gag aat gca aaa aaa gct att 432 Gln Leu Gly Ile Glu AsnVal Thr Asp Glu Asn Ala Lys Lys Ala Ile 130 135 140 tta ata aca gat gcagct aaa gat aag ggc gct gca gag ctt gaa aag 480 Leu Ile Thr Asp Ala AlaLys Asp Lys Gly Ala Ala Glu Leu Glu Lys 145 150 155 160 cta ttt aaa gcagta gaa aac ttg gca aaa gca gct aaa gag atg ctt 528 Leu Phe Lys Ala ValGlu Asn Leu Ala Lys Ala Ala Lys Glu Met Leu 165 170 175 gct aat tca gttaaa gag ctt aca agt cct att gtg cat ggc gtt tca 576 Ala Asn Ser Val LysGlu Leu Thr Ser Pro Ile Val His Gly Val Ser 180 185 190 gta gat ttg cctggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac 624 Val Asp Leu Pro GlyGlu Met Lys Val Leu Val Ser Lys Glu Lys Asn 195 200 205 aaa gac ggc aagtac gat cta att gca aca gta gac aag ctt gag ctt 672 Lys Asp Gly Lys TyrAsp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu 210 215 220 aaa gga act tctgat aaa aac aat gga tct gga gta ctt gaa ggc gta 720 Lys Gly Thr Ser AspLys Asn Asn Gly Ser Gly Val Leu Glu Gly Val 225 230 235 240 aaa gct gacaaa agt aaa gta aaa tta aca att tct gac gat cta ggt 768 Lys Ala Asp LysSer Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly 245 250 255 caa acc acactt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca 816 Gln Thr Thr LeuGlu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser 260 265 270 aaa aaa gtaact tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat 864 Lys Lys Val ThrSer Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn 275 280 285 gaa aaa ggtgaa gta tct gaa aaa ata ata aca aga gca gac gga acc 912 Glu Lys Gly GluVal Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr 290 295 300 aga ctt gaatac aca gga att aaa agc gat gga tct gga aaa gct aaa 960 Arg Leu Glu TyrThr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys 305 310 315 320 gag gtttta aaa aaa ttt act ctt gaa gga aaa gta gct aat gat aaa 1008 Glu Val LeuLys Lys Phe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys 325 330 335 gta acattg gaa gta aaa gaa gga acc gtt act tta agt aag aac att 1056 Val Thr LeuGlu Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile 340 345 350 tca aaatct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt 1104 Ser Lys SerGly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser 355 360 365 gct gctact aaa aaa act gca gct tgg aat tca ggc act tca act tta 1152 Ala Ala ThrLys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu 370 375 380 aca attact gta aac agt aaa aaa act aaa gac ctt gtg ttt aca aaa 1200 Thr Ile ThrVal Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys 385 390 395 400 gaaaac aca att aca gta caa caa tac gac tca aat ggc acc aaa tta 1248 Glu AsnThr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu 405 410 415 gagggg tca gca gtt gaa att aca aaa ctt gat gaa att aaa aac gct 1296 Glu GlySer Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala 420 425 430 ttaaaa taa 1305 Leu Lys * 74 434 PRT Artificial Sequence Chimeric protein74 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 510 15 Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 2025 30 Ile Thr Glu Ser Asn Ala Val Val Leu Ala Val Lys Glu Ile Glu Thr 3540 45 Leu Leu Ala Ser Ile Asp Glu Leu Ala Thr Lys Ala Ile Gly Lys Lys 5055 60 Ile Gln Gln Asn Gly Gly Leu Ala Val Glu Ala Gly His Asn Gly Thr 6570 75 80 Leu Leu Ala Gly Ala Tyr Thr Ile Ser Lys Leu Ile Thr Gln Lys Leu85 90 95 Asp Gly Leu Lys Asn Ser Glu Lys Leu Lys Glu Lys Ile Glu Asn Ala100 105 110 Lys Lys Cys Ser Glu Asp Phe Thr Lys Lys Leu Glu Gly Glu HisAla 115 120 125 Gln Leu Gly Ile Glu Asn Val Thr Asp Glu Asn Ala Lys LysAla Ile 130 135 140 Leu Ile Thr Asp Ala Ala Lys Asp Lys Gly Ala Ala GluLeu Glu Lys 145 150 155 160 Leu Phe Lys Ala Val Glu Asn Leu Ala Lys AlaAla Lys Glu Met Leu 165 170 175 Ala Asn Ser Val Lys Glu Leu Thr Ser ProIle Val His Gly Val Ser 180 185 190 Val Asp Leu Pro Gly Glu Met Lys ValLeu Val Ser Lys Glu Lys Asn 195 200 205 Lys Asp Gly Lys Tyr Asp Leu IleAla Thr Val Asp Lys Leu Glu Leu 210 215 220 Lys Gly Thr Ser Asp Lys AsnAsn Gly Ser Gly Val Leu Glu Gly Val 225 230 235 240 Lys Ala Asp Lys SerLys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly 245 250 255 Gln Thr Thr LeuGlu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser 260 265 270 Lys Lys ValThr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn 275 280 285 Glu LysGly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr 290 295 300 ArgLeu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys 305 310 315320 Glu Val Leu Lys Lys Phe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys 325330 335 Val Thr Leu Glu Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile340 345 350 Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp SerSer 355 360 365 Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr SerThr Leu 370 375 380 Thr Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu ValPhe Thr Lys 385 390 395 400 Glu Asn Thr Ile Thr Val Gln Gln Tyr Asp SerAsn Gly Thr Lys Leu 405 410 415 Glu Gly Ser Ala Val Glu Ile Thr Lys LeuAsp Glu Ile Lys Asn Ala 420 425 430 Leu Lys 75 1332 DNA ArtificialSequence Chimeric nucleic acid 75 atg gct tgt agt aat tca ggg aaa ggtggg gat tct gca tct act aat 48 Met Ala Cys Ser Asn Ser Gly Lys Gly GlyAsp Ser Ala Ser Thr Asn 1 5 10 15 cct gct gac gag tct gcg aaa ggg cctaat ctt aca gaa ata agc aaa 96 Pro Ala Asp Glu Ser Ala Lys Gly Pro AsnLeu Thr Glu Ile Ser Lys 20 25 30 aaa att aca gat tct aat gca ttt gta cttgct gtt aaa gaa gtt gag 144 Lys Ile Thr Asp Ser Asn Ala Phe Val Leu AlaVal Lys Glu Val Glu 35 40 45 act ttg gtt tta tct ata gat gaa ctt gct aagaaa gct att ggt caa 192 Thr Leu Val Leu Ser Ile Asp Glu Leu Ala Lys LysAla Ile Gly Gln 50 55 60 aaa ata gac aat aat aat ggt tta gct gct tta aataat cag aat gga 240 Lys Ile Asp Asn Asn Asn Gly Leu Ala Ala Leu Asn AsnGln Asn Gly 65 70 75 80 tcg ttg tta gca gga gcc tat gca ata tca acc ctaata aca gaa aaa 288 Ser Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu IleThr Glu Lys 85 90 95 ttg agt aaa ttg aaa aat tta gaa gaa tta aag aca gaaatt gca aag 336 Leu Ser Lys Leu Lys Asn Leu Glu Glu Leu Lys Thr Glu IleAla Lys 100 105 110 gct aag aaa tgt tcc gaa gaa ttt act aat aaa cta aaaagt ggt cat 384 Ala Lys Lys Cys Ser Glu Glu Phe Thr Asn Lys Leu Lys SerGly His 115 120 125 gca gat ctt ggc aaa cag gat gct acc gat gat cat gcaaaa gca gct 432 Ala Asp Leu Gly Lys Gln Asp Ala Thr Asp Asp His Ala LysAla Ala 130 135 140 att tta aaa aca cat gca act acc gat aaa ggt gct aaagaa ttt aaa 480 Ile Leu Lys Thr His Ala Thr Thr Asp Lys Gly Ala Lys GluPhe Lys 145 150 155 160 gat tta ttt gaa tca gta gaa ggt ttg tta aaa gcagct caa gta gca 528 Asp Leu Phe Glu Ser Val Glu Gly Leu Leu Lys Ala AlaGln Val Ala 165 170 175 cta act aat tca gtt aaa gaa ctt aca agt cct gttgta gca gaa agt 576 Leu Thr Asn Ser Val Lys Glu Leu Thr Ser Pro Val ValAla Glu Ser 180 185 190 cca aaa aaa cct tcc atg gcc gtt tca gta gat ttgcct ggt gaa atg 624 Pro Lys Lys Pro Ser Met Ala Val Ser Val Asp Leu ProGly Glu Met 195 200 205 aaa gtt ctt gta agc aaa gaa aaa aac aaa gac ggcaag tac gat cta 672 Lys Val Leu Val Ser Lys Glu Lys Asn Lys Asp Gly LysTyr Asp Leu 210 215 220 att gca aca gta gac aag ctt gag ctt aaa gga acttct gat aaa aac 720 Ile Ala Thr Val Asp Lys Leu Glu Leu Lys Gly Thr SerAsp Lys Asn 225 230 235 240 aat gga tct gga gta ctt gaa ggc gta aaa gctgac aaa agt aaa gta 768 Asn Gly Ser Gly Val Leu Glu Gly Val Lys Ala AspLys Ser Lys Val 245 250 255 aaa tta aca att tct gac gat cta ggt caa accaca ctt gaa gtt ttc 816 Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln Thr ThrLeu Glu Val Phe 260 265 270 aaa gaa gat ggc aaa aca cta gta tca aaa aaagta act tcc aaa gac 864 Lys Glu Asp Gly Lys Thr Leu Val Ser Lys Lys ValThr Ser Lys Asp 275 280 285 aag tca tca aca gaa gaa aaa ttc aat gaa aaaggt gaa gta tct gaa 912 Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu Lys GlyGlu Val Ser Glu 290 295 300 aaa ata ata aca aga gca gac gga acc aga cttgaa tac aca gga att 960 Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg Leu GluTyr Thr Gly Ile 305 310 315 320 aaa agc gat gga tct gga aaa gct aaa gaggtt tta aaa aaa ttt act 1008 Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu ValLeu Lys Lys Phe Thr 325 330 335 ctt gaa gga aaa gta gct aat gat aaa gtaaca ttg gaa gta aaa gaa 1056 Leu Glu Gly Lys Val Ala Asn Asp Lys Val ThrLeu Glu Val Lys Glu 340 345 350 gga acc gtt act tta agt aag aat att tcaaaa tct ggg gaa gtt tca 1104 Gly Thr Val Thr Leu Ser Lys Asn Ile Ser LysSer Gly Glu Val Ser 355 360 365 gtt gaa ctt aat gac act gac agt agt gctgct act aaa aaa act gca 1152 Val Glu Leu Asn Asp Thr Asp Ser Ser Ala AlaThr Lys Lys Thr Ala 370 375 380 gct tgg aat tca aaa act tcc act tta acaatt agt gtg aat agc caa 1200 Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr IleSer Val Asn Ser Gln 385 390 395 400 aaa acc aaa aac ctt gta ttc aca aaagaa gac aca ata aca gta caa 1248 Lys Thr Lys Asn Leu Val Phe Thr Lys GluAsp Thr Ile Thr Val Gln 405 410 415 aaa tac gac tca gca ggc acc aat ctagaa ggc aaa gca gtc gaa att 1296 Lys Tyr Asp Ser Ala Gly Thr Asn Leu GluGly Lys Ala Val Glu Ile 420 425 430 aca aca ctt aaa gaa ctt aaa aac gcttta aaa taa 1332 Thr Thr Leu Lys Glu Leu Lys Asn Ala Leu Lys * 435 44076 443 PRT Artificial Sequence Chimeric protein 76 Met Ala Cys Ser AsnSer Gly Lys Gly Gly Asp Ser Ala Ser Thr Asn 1 5 10 15 Pro Ala Asp GluSer Ala Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys 20 25 30 Lys Ile Thr AspSer Asn Ala Phe Val Leu Ala Val Lys Glu Val Glu 35 40 45 Thr Leu Val LeuSer Ile Asp Glu Leu Ala Lys Lys Ala Ile Gly Gln 50 55 60 Lys Ile Asp AsnAsn Asn Gly Leu Ala Ala Leu Asn Asn Gln Asn Gly 65 70 75 80 Ser Leu LeuAla Gly Ala Tyr Ala Ile Ser Thr Leu Ile Thr Glu Lys 85 90 95 Leu Ser LysLeu Lys Asn Leu Glu Glu Leu Lys Thr Glu Ile Ala Lys 100 105 110 Ala LysLys Cys Ser Glu Glu Phe Thr Asn Lys Leu Lys Ser Gly His 115 120 125 AlaAsp Leu Gly Lys Gln Asp Ala Thr Asp Asp His Ala Lys Ala Ala 130 135 140Ile Leu Lys Thr His Ala Thr Thr Asp Lys Gly Ala Lys Glu Phe Lys 145 150155 160 Asp Leu Phe Glu Ser Val Glu Gly Leu Leu Lys Ala Ala Gln Val Ala165 170 175 Leu Thr Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala GluSer 180 185 190 Pro Lys Lys Pro Ser Met Ala Val Ser Val Asp Leu Pro GlyGlu Met 195 200 205 Lys Val Leu Val Ser Lys Glu Lys Asn Lys Asp Gly LysTyr Asp Leu 210 215 220 Ile Ala Thr Val Asp Lys Leu Glu Leu Lys Gly ThrSer Asp Lys Asn 225 230 235 240 Asn Gly Ser Gly Val Leu Glu Gly Val LysAla Asp Lys Ser Lys Val 245 250 255 Lys Leu Thr Ile Ser Asp Asp Leu GlyGln Thr Thr Leu Glu Val Phe 260 265 270 Lys Glu Asp Gly Lys Thr Leu ValSer Lys Lys Val Thr Ser Lys Asp 275 280 285 Lys Ser Ser Thr Glu Glu LysPhe Asn Glu Lys Gly Glu Val Ser Glu 290 295 300 Lys Ile Ile Thr Arg AlaAsp Gly Thr Arg Leu Glu Tyr Thr Gly Ile 305 310 315 320 Lys Ser Asp GlySer Gly Lys Ala Lys Glu Val Leu Lys Lys Phe Thr 325 330 335 Leu Glu GlyLys Val Ala Asn Asp Lys Val Thr Leu Glu Val Lys Glu 340 345 350 Gly ThrVal Thr Leu Ser Lys Asn Ile Ser Lys Ser Gly Glu Val Ser 355 360 365 ValGlu Leu Asn Asp Thr Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala 370 375 380Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr Ile Ser Val Asn Ser Gln 385 390395 400 Lys Thr Lys Asn Leu Val Phe Thr Lys Glu Asp Thr Ile Thr Val Gln405 410 415 Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu Gly Lys Ala Val GluIle 420 425 430 Thr Thr Leu Lys Glu Leu Lys Asn Ala Leu Lys 435 440 771317 DNA Artificial Sequence Chimeric nucleic acid 77 atg gct tgt aataat tca ggt ggg gat tct gca tct act aat cct gat 48 Met Ala Cys Asn AsnSer Gly Gly Asp Ser Ala Ser Thr Asn Pro Asp 1 5 10 15 gag tct gca aaagga cct aat ctt acc gta ata agc aaa aaa att aca 96 Glu Ser Ala Lys GlyPro Asn Leu Thr Val Ile Ser Lys Lys Ile Thr 20 25 30 gat tct aat gca ttttta ctg gct gtg aaa gaa gtt gag gct ttg ctt 144 Asp Ser Asn Ala Phe LeuLeu Ala Val Lys Glu Val Glu Ala Leu Leu 35 40 45 tca tct ata gat gaa ctttct aaa gct att ggt aaa aaa ata aaa aat 192 Ser Ser Ile Asp Glu Leu SerLys Ala Ile Gly Lys Lys Ile Lys Asn 50 55 60 gat ggt act tta gat aac gaagca aat cga aac gaa tca ttg ata gca 240 Asp Gly Thr Leu Asp Asn Glu AlaAsn Arg Asn Glu Ser Leu Ile Ala 65 70 75 80 gga gct tat gaa ata tca aaacta ata aca caa aaa tta agt gta ttg 288 Gly Ala Tyr Glu Ile Ser Lys LeuIle Thr Gln Lys Leu Ser Val Leu 85 90 95 aat tca gaa gaa tta aag gaa aaaatt aaa gag gct aag gat tgt tcc 336 Asn Ser Glu Glu Leu Lys Glu Lys IleLys Glu Ala Lys Asp Cys Ser 100 105 110 gaa aaa ttt act act aag cta aaagat agt cat gca gag ctt ggt ata 384 Glu Lys Phe Thr Thr Lys Leu Lys AspSer His Ala Glu Leu Gly Ile 115 120 125 caa agc gtt cag gat gat aat gcaaaa aaa gct att tta aaa aca cat 432 Gln Ser Val Gln Asp Asp Asn Ala LysLys Ala Ile Leu Lys Thr His 130 135 140 gga act aaa gac aag ggt gct aaagaa ctt gaa gag tta ttt aaa tca 480 Gly Thr Lys Asp Lys Gly Ala Lys GluLeu Glu Glu Leu Phe Lys Ser 145 150 155 160 cta gaa agc ttg tca aaa gcagcg caa gca gca tta act aat tca gtt 528 Leu Glu Ser Leu Ser Lys Ala AlaGln Ala Ala Leu Thr Asn Ser Val 165 170 175 aaa gag ctt aca aat cct gttgtg gca gaa agt cca aaa aaa cct tcc 576 Lys Glu Leu Thr Asn Pro Val ValAla Glu Ser Pro Lys Lys Pro Ser 180 185 190 atg gcc gtt tca gta gat ttgcct ggt gaa atg aaa gtt ctt gta agc 624 Met Ala Val Ser Val Asp Leu ProGly Glu Met Lys Val Leu Val Ser 195 200 205 aaa gaa aaa aac aaa gac ggcaag tac gat cta att gca aca gta gac 672 Lys Glu Lys Asn Lys Asp Gly LysTyr Asp Leu Ile Ala Thr Val Asp 210 215 220 aag ctt gag ctt aaa gga acttct gat aaa aac aat gga tct gga gta 720 Lys Leu Glu Leu Lys Gly Thr SerAsp Lys Asn Asn Gly Ser Gly Val 225 230 235 240 ctt gaa ggc gta aaa gctgac aaa agt aaa gta aaa tta aca att tct 768 Leu Glu Gly Val Lys Ala AspLys Ser Lys Val Lys Leu Thr Ile Ser 245 250 255 gac gat cta ggt caa accaca ctt gaa gtt ttc aaa gaa gat ggc aaa 816 Asp Asp Leu Gly Gln Thr ThrLeu Glu Val Phe Lys Glu Asp Gly Lys 260 265 270 aca cta gta tca aaa aaagta act tcc aaa gac aag tca tca aca gaa 864 Thr Leu Val Ser Lys Lys ValThr Ser Lys Asp Lys Ser Ser Thr Glu 275 280 285 gaa aaa ttc aat gaa aaaggt gaa gta tct gaa aaa ata ata aca aga 912 Glu Lys Phe Asn Glu Lys GlyGlu Val Ser Glu Lys Ile Ile Thr Arg 290 295 300 gca gac gga acc aga cttgaa tac aca gga att aaa agc gat gga tct 960 Ala Asp Gly Thr Arg Leu GluTyr Thr Gly Ile Lys Ser Asp Gly Ser 305 310 315 320 gga aaa gct aaa gaggtt tta aaa aaa ttt act ctt gaa gga aaa gta 1008 Gly Lys Ala Lys Glu ValLeu Lys Lys Phe Thr Leu Glu Gly Lys Val 325 330 335 gct aat gat aaa gtaaca ttg gaa gta aaa gaa gga acc gtt act tta 1056 Ala Asn Asp Lys Val ThrLeu Glu Val Lys Glu Gly Thr Val Thr Leu 340 345 350 agt aag aac att tcaaaa tct ggg gaa gtt tca gtt gaa ctt aat gac 1104 Ser Lys Asn Ile Ser LysSer Gly Glu Val Ser Val Glu Leu Asn Asp 355 360 365 act gac agt agt gctgct act aaa aaa act gca gct tgg aat tca aaa 1152 Thr Asp Ser Ser Ala AlaThr Lys Lys Thr Ala Ala Trp Asn Ser Lys 370 375 380 act tct act tta acaatt agt gtt aac agc aaa aaa act aca caa ctt 1200 Thr Ser Thr Leu Thr IleSer Val Asn Ser Lys Lys Thr Thr Gln Leu 385 390 395 400 gtg ttt act aaacaa gac aca ata act gta caa aaa tac gac tcc gca 1248 Val Phe Thr Lys GlnAsp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala 405 410 415 ggt acc aat ttagaa ggc aca gca gtc gaa att aaa aca ctt gat gaa 1296 Gly Thr Asn Leu GluGly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu 420 425 430 ctt aaa aac gcttta aaa taa 1317 Leu Lys Asn Ala Leu Lys * 435 78 438 PRT ArtificialSequence Chimeric protein 78 Met Ala Cys Asn Asn Ser Gly Gly Asp Ser AlaSer Thr Asn Pro Asp 1 5 10 15 Glu Ser Ala Lys Gly Pro Asn Leu Thr ValIle Ser Lys Lys Ile Thr 20 25 30 Asp Ser Asn Ala Phe Leu Leu Ala Val LysGlu Val Glu Ala Leu Leu 35 40 45 Ser Ser Ile Asp Glu Leu Ser Lys Ala IleGly Lys Lys Ile Lys Asn 50 55 60 Asp Gly Thr Leu Asp Asn Glu Ala Asn ArgAsn Glu Ser Leu Ile Ala 65 70 75 80 Gly Ala Tyr Glu Ile Ser Lys Leu IleThr Gln Lys Leu Ser Val Leu 85 90 95 Asn Ser Glu Glu Leu Lys Glu Lys IleLys Glu Ala Lys Asp Cys Ser 100 105 110 Glu Lys Phe Thr Thr Lys Leu LysAsp Ser His Ala Glu Leu Gly Ile 115 120 125 Gln Ser Val Gln Asp Asp AsnAla Lys Lys Ala Ile Leu Lys Thr His 130 135 140 Gly Thr Lys Asp Lys GlyAla Lys Glu Leu Glu Glu Leu Phe Lys Ser 145 150 155 160 Leu Glu Ser LeuSer Lys Ala Ala Gln Ala Ala Leu Thr Asn Ser Val 165 170 175 Lys Glu LeuThr Asn Pro Val Val Ala Glu Ser Pro Lys Lys Pro Ser 180 185 190 Met AlaVal Ser Val Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser 195 200 205 LysGlu Lys Asn Lys Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp 210 215 220Lys Leu Glu Leu Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val 225 230235 240 Leu Glu Gly Val Lys Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser245 250 255 Asp Asp Leu Gly Gln Thr Thr Leu Glu Val Phe Lys Glu Asp GlyLys 260 265 270 Thr Leu Val Ser Lys Lys Val Thr Ser Lys Asp Lys Ser SerThr Glu 275 280 285 Glu Lys Phe Asn Glu Lys Gly Glu Val Ser Glu Lys IleIle Thr Arg 290 295 300 Ala Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile LysSer Asp Gly Ser 305 310 315 320 Gly Lys Ala Lys Glu Val Leu Lys Lys PheThr Leu Glu Gly Lys Val 325 330 335 Ala Asn Asp Lys Val Thr Leu Glu ValLys Glu Gly Thr Val Thr Leu 340 345 350 Ser Lys Asn Ile Ser Lys Ser GlyGlu Val Ser Val Glu Leu Asn Asp 355 360 365 Thr Asp Ser Ser Ala Ala ThrLys Lys Thr Ala Ala Trp Asn Ser Lys 370 375 380 Thr Ser Thr Leu Thr IleSer Val Asn Ser Lys Lys Thr Thr Gln Leu 385 390 395 400 Val Phe Thr LysGln Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala 405 410 415 Gly Thr AsnLeu Glu Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu 420 425 430 Leu LysAsn Ala Leu Lys 435 79 1029 DNA Artificial Sequence Chimeric nucleicacid 79 atg gct tgt aat aat tca ggg aaa gat ggg aat aca tct gca aat tct48 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 510 15 gct gat gag tct gtt aaa ggg cct aat ctt aca gaa ata aat aaa aaa 96Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30att acg gat tct aat gcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 IleThr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttgctg tca tct ata gat gaa att gct gct aaa gct att ggt aaa aaa 192 Leu LeuSer Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 ata caccaa aat aat ggt ttg gat acc gaa aat aat cac aat gga tca 240 Ile His GlnAsn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 ttg ttagcg gga gct tat gca ata tca acc cta ata aaa caa aaa tta 288 Leu Leu AlaGly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 gat gga ttgaaa aat gaa gga tta aag gaa aaa att gat gcg gct aag 336 Asp Gly Leu LysAsn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 aaa tgt tctgaa aca ttt act aat aaa tta aaa gaa aaa cac aca gat 384 Lys Cys Ser GluThr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 ctt ggt aaagaa ggt gtt act gat gct gat gca aaa gaa gcc att tta 432 Leu Gly Lys GluGly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 aaa gca aatggt act aaa act aaa ggt gct gaa gaa ctt gga aaa tta 480 Lys Ala Asn GlyThr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 ttt gaatca gta gag gtc ttg tca aaa gca gct aaa gag atg ctt gct 528 Phe Glu SerVal Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 aat tcagtt aaa gag ctt aca agc cct gtt gtg gca gaa agt cca aaa 576 Asn Ser ValLys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 aaa ccttcc atg gcc aag caa aat gtt tct gaa aaa ata ata aca aga 624 Lys Pro SerMet Ala Lys Gln Asn Val Ser Glu Lys Ile Ile Thr Arg 195 200 205 gca gacgga acc aga ctt gaa tac aca gga att aaa agc gat gga tct 672 Ala Asp GlyThr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser 210 215 220 gga aaagct aaa gag gtt tta aaa ggc tat gtt ctt gaa gga act cta 720 Gly Lys AlaLys Glu Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu 225 230 235 240 actgct gaa aaa aca aca ttg gtg gtt aaa gaa gga act gtt act tta 768 Thr AlaGlu Lys Thr Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu 245 250 255 agcaaa aat att tca aaa tct ggg gaa gtt tca gtt gaa ctt aat gac 816 Ser LysAsn Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp 260 265 270 actgac agt agt gct gct act aaa aaa act gca gct tgg aat tca ggc 864 Thr AspSer Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly 275 280 285 acttca act tta aca att act gta aac agt aaa aaa act aaa gac ctt 912 Thr SerThr Leu Thr Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu 290 295 300 gtgttt aca aaa gaa aac aca att aca gta caa caa tac gac tca aat 960 Val PheThr Lys Glu Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn 305 310 315 320ggc acc aaa tta gag ggg tca gca gtt gaa att aca aaa ctt gat gaa 1008 GlyThr Lys Leu Glu Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu 325 330 335att aaa aac gct tta aaa taa 1029 Ile Lys Asn Ala Leu Lys * 340 80 342PRT Artificial Sequence Chimeric protein 80 Met Ala Cys Asn Asn Ser GlyLys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser Val LysGly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 Ile Thr Asp Ser Asn AlaVal Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu Ser Ser Ile AspGlu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 Ile His Gln Asn Asn GlyLeu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 Leu Leu Ala Gly AlaTyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 Asp Gly Leu Lys AsnGlu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 Lys Cys Ser GluThr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 Leu Gly LysGlu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 Lys AlaAsn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170175 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180185 190 Lys Pro Ser Met Ala Lys Gln Asn Val Ser Glu Lys Ile Ile Thr Arg195 200 205 Ala Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp GlySer 210 215 220 Gly Lys Ala Lys Glu Val Leu Lys Gly Tyr Val Leu Glu GlyThr Leu 225 230 235 240 Thr Ala Glu Lys Thr Thr Leu Val Val Lys Glu GlyThr Val Thr Leu 245 250 255 Ser Lys Asn Ile Ser Lys Ser Gly Glu Val SerVal Glu Leu Asn Asp 260 265 270 Thr Asp Ser Ser Ala Ala Thr Lys Lys ThrAla Ala Trp Asn Ser Gly 275 280 285 Thr Ser Thr Leu Thr Ile Thr Val AsnSer Lys Lys Thr Lys Asp Leu 290 295 300 Val Phe Thr Lys Glu Asn Thr IleThr Val Gln Gln Tyr Asp Ser Asn 305 310 315 320 Gly Thr Lys Leu Glu GlySer Ala Val Glu Ile Thr Lys Leu Asp Glu 325 330 335 Ile Lys Asn Ala LeuLys 340 82 342 PRT Artificial Sequence Chimeric protein 82 Met Ala CysAsn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala AspGlu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 Ile ThrAsp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu LeuSer Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 Ile HisGln Asn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 LeuLeu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 AspGly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120125 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130135 140 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu145 150 155 160 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu MetLeu Ala 165 170 175 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala GluSer Pro Lys 180 185 190 Lys Pro Ser Met Ala Lys Gln Asn Val Ser Glu LysIle Ile Thr Arg 195 200 205 Ala Asp Gly Thr Arg Leu Glu Tyr Thr Gly IleLys Ser Asp Gly Ser 210 215 220 Gly Lys Ala Lys Glu Val Leu Lys Gly TyrVal Leu Glu Gly Thr Leu 225 230 235 240 Thr Ala Glu Lys Thr Thr Leu ValVal Lys Glu Gly Thr Val Thr Leu 245 250 255 Ser Lys Asn Ile Ser Lys SerGly Glu Val Ser Val Glu Leu Asn Asp 260 265 270 Thr Asp Ser Ser Ala AlaThr Lys Lys Thr Ala Ala Trp Asn Ser Lys 275 280 285 Thr Ser Thr Leu ThrIle Ser Val Asn Ser Gln Lys Thr Lys Asn Leu 290 295 300 Val Phe Thr LysGlu Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala 305 310 315 320 Gly ThrAsn Leu Glu Gly Lys Ala Val Glu Ile Thr Thr Leu Lys Glu 325 330 335 LeuLys Asn Ala Leu Lys 340 83 1029 DNA Artificial Sequence Chimeric nucleicacid 83 atg gct tgt aat aat tca ggg aaa gat ggg aat aca tct gca aat tct48 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 510 15 gct gat gag tct gtt aaa ggg cct aat ctt aca gaa ata aat aaa aaa 96Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30att acg gat tct aat gcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 IleThr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttgctg tca tct ata gat gaa att gct gct aaa gct att ggt aaa aaa 192 Leu LeuSer Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 ata caccaa aat aat ggt ttg gat acc gaa aat aat cac aat gga tca 240 Ile His GlnAsn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 ttg ttagcg gga gct tat gca ata tca acc cta ata aaa caa aaa tta 288 Leu Leu AlaGly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 gat gga ttgaaa aat gaa gga tta aag gaa aaa att gat gcg gct aag 336 Asp Gly Leu LysAsn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 aaa tgt tctgaa aca ttt act aat aaa tta aaa gaa aaa cac aca gat 384 Lys Cys Ser GluThr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 ctt ggt aaagaa ggt gtt act gat gct gat gca aaa gaa gcc att tta 432 Leu Gly Lys GluGly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 aaa gca aatggt act aaa act aaa ggt gct gaa gaa ctt gga aaa tta 480 Lys Ala Asn GlyThr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 ttt gaatca gta gag gtc ttg tca aaa gca gct aaa gag atg ctt gct 528 Phe Glu SerVal Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 aat tcagtt aaa gag ctt aca agc cct gtt gtg gca gaa agt cca aaa 576 Asn Ser ValLys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 aaa ccttcc atg gcc aag caa aat gtt tct gaa aaa ata ata aca aga 624 Lys Pro SerMet Ala Lys Gln Asn Val Ser Glu Lys Ile Ile Thr Arg 195 200 205 gca gacgga acc aga ctt gaa tac aca gga att aaa agc gat gga tct 672 Ala Asp GlyThr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser 210 215 220 gga aaagct aaa gag gtt tta aaa ggc tat gtt ctt gaa gga act cta 720 Gly Lys AlaLys Glu Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu 225 230 235 240 actgct gaa aaa aca aca ttg gtg gtt aaa gaa gga act gtt act tta 768 Thr AlaGlu Lys Thr Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu 245 250 255 agcaaa aat att tca aaa tct ggg gaa gtt tca gtt gaa ctt aat gac 816 Ser LysAsn Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp 260 265 270 actgac agt agt gct gct act aaa aaa act gca gct tgg aat tca aaa 864 Thr AspSer Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys 275 280 285 acttct act tta aca att agt gtt aac agc aaa aaa act aca caa ctt 912 Thr SerThr Leu Thr Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu 290 295 300 gtgttt act aaa caa gac aca ata act gta caa aaa tac gac tcc gca 960 Val PheThr Lys Gln Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala 305 310 315 320ggt acc aat tta gaa ggc aca gca gtc gaa att aaa aca ctt gat gaa 1008 GlyThr Asn Leu Glu Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu 325 330 335ctt aaa aac gct tta aaa taa 1029 Leu Lys Asn Ala Leu Lys * 340 84 342PRT Artificial Sequence Chimeric protein 84 Met Ala Cys Asn Asn Ser GlyLys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser Val LysGly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 Ile Thr Asp Ser Asn AlaVal Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu Ser Ser Ile AspGlu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 Ile His Gln Asn Asn GlyLeu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 Leu Leu Ala Gly AlaTyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 Asp Gly Leu Lys AsnGlu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 Lys Cys Ser GluThr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 Leu Gly LysGlu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 Lys AlaAsn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170175 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180185 190 Lys Pro Ser Met Ala Lys Gln Asn Val Ser Glu Lys Ile Ile Thr Arg195 200 205 Ala Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser Asp GlySer 210 215 220 Gly Lys Ala Lys Glu Val Leu Lys Gly Tyr Val Leu Glu GlyThr Leu 225 230 235 240 Thr Ala Glu Lys Thr Thr Leu Val Val Lys Glu GlyThr Val Thr Leu 245 250 255 Ser Lys Asn Ile Ser Lys Ser Gly Glu Val SerVal Glu Leu Asn Asp 260 265 270 Thr Asp Ser Ser Ala Ala Thr Lys Lys ThrAla Ala Trp Asn Ser Lys 275 280 285 Thr Ser Thr Leu Thr Ile Ser Val AsnSer Lys Lys Thr Thr Gln Leu 290 295 300 Val Phe Thr Lys Gln Asp Thr IleThr Val Gln Lys Tyr Asp Ser Ala 305 310 315 320 Gly Thr Asn Leu Glu GlyThr Ala Val Glu Ile Lys Thr Leu Asp Glu 325 330 335 Leu Lys Asn Ala LeuLys 340 85 1035 DNA Artificial Sequence Chimeric nucleic acid 85 atg gcttgt aat aat tca ggg aaa gat ggg aat aca tct gca aat tct 48 Met Ala CysAsn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gatgag tct gtt aaa ggg cct aat ctt aca gaa ata agt aaa aaa 96 Ala Asp GluSer Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 att acg gattct aat gcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Asp SerAsn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttg ctg tca tctata gat gaa att gct gct aaa gct att ggt aaa aaa 192 Leu Leu Ser Ser IleAsp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 ata cac caa aat aatggt ttg gat acc gaa tat aat cac aat gga tca 240 Ile His Gln Asn Asn GlyLeu Asp Thr Glu Tyr Asn His Asn Gly Ser 65 70 75 80 ttg tta gcg gga gcttat gca ata tca acc cta ata aaa caa aaa tta 288 Leu Leu Ala Gly Ala TyrAla Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 gat gga ttg aaa aat gaagga tta aag gaa aaa att gat gcg gct aag 336 Asp Gly Leu Lys Asn Glu GlyLeu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 aaa tgt tct gaa aca tttact aat aaa tta aaa gaa aaa cac aca gat 384 Lys Cys Ser Glu Thr Phe ThrAsn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 ctt ggt aaa gaa ggt gttact gat gct gat gca aaa gaa gcc att tta 432 Leu Gly Lys Glu Gly Val ThrAsp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 aaa aca aat ggt act aaaact aaa ggt gct gaa gaa ctt gga aaa tta 480 Lys Thr Asn Gly Thr Lys ThrLys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 ttt gaa tca gta gaggtc ttg tca aaa gca gct aaa gag atg ctt gct 528 Phe Glu Ser Val Glu ValLeu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 aat tca gtt aaa gagctt aca agc cct gtt gtg gca gaa agt cca aaa 576 Asn Ser Val Lys Glu LeuThr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 aaa cct tcc atg gccaag caa aat gtt aca tct gaa aaa aca ata gta 624 Lys Pro Ser Met Ala LysGln Asn Val Thr Ser Glu Lys Thr Ile Val 195 200 205 aga gca aat gga accaga ctt gaa tac aca gac ata aaa agc gat gga 672 Arg Ala Asn Gly Thr ArgLeu Glu Tyr Thr Asp Ile Lys Ser Asp Gly 210 215 220 tcc gga aaa gct aaagaa gtt tta aaa gac ttt act ctt gaa gga act 720 Ser Gly Lys Ala Lys GluVal Leu Lys Asp Phe Thr Leu Glu Gly Thr 225 230 235 240 cta gct gct gacggc aaa aca aca ttg aaa gtt aca gaa ggc act gtt 768 Leu Ala Ala Asp GlyLys Thr Thr Leu Lys Val Thr Glu Gly Thr Val 245 250 255 gtt tta agc aagaac att tta aaa tcc gga gaa ata aca gtt gca ctt 816 Val Leu Ser Lys AsnIle Leu Lys Ser Gly Glu Ile Thr Val Ala Leu 260 265 270 gat gac tct gacact act cag gct act aaa aaa act gga aaa tgg gat 864 Asp Asp Ser Asp ThrThr Gln Ala Thr Lys Lys Thr Gly Lys Trp Asp 275 280 285 tca aat act tccact tta aca att agt gtg aat agc aaa aaa act aaa 912 Ser Asn Thr Ser ThrLeu Thr Ile Ser Val Asn Ser Lys Lys Thr Lys 290 295 300 aac att gta tttaca aaa gaa gac aca ata aca gta caa aaa tac gac 960 Asn Ile Val Phe ThrLys Glu Asp Thr Ile Thr Val Gln Lys Tyr Asp 305 310 315 320 tca gca ggcacc aat cta gaa ggc aac gca gtc gaa att aaa aca ctt 1008 Ser Ala Gly ThrAsn Leu Glu Gly Asn Ala Val Glu Ile Lys Thr Leu 325 330 335 gat gaa cttaaa aac gct tta aaa tag 1035 Asp Glu Leu Lys Asn Ala Leu Lys * 340 86344 PRT Artificial Sequence Chimeric protein 86 Met Ala Cys Asn Asn SerGly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp Glu Ser ValLys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 Ile Thr Asp Ser AsnAla Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu Ser Ser IleAsp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 Ile His Gln Asn AsnGly Leu Asp Thr Glu Tyr Asn His Asn Gly Ser 65 70 75 80 Leu Leu Ala GlyAla Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 Asp Gly Leu LysAsn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 Lys Cys SerGlu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 Leu GlyLys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 LysThr Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155160 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165170 175 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys180 185 190 Lys Pro Ser Met Ala Lys Gln Asn Val Thr Ser Glu Lys Thr IleVal 195 200 205 Arg Ala Asn Gly Thr Arg Leu Glu Tyr Thr Asp Ile Lys SerAsp Gly 210 215 220 Ser Gly Lys Ala Lys Glu Val Leu Lys Asp Phe Thr LeuGlu Gly Thr 225 230 235 240 Leu Ala Ala Asp Gly Lys Thr Thr Leu Lys ValThr Glu Gly Thr Val 245 250 255 Val Leu Ser Lys Asn Ile Leu Lys Ser GlyGlu Ile Thr Val Ala Leu 260 265 270 Asp Asp Ser Asp Thr Thr Gln Ala ThrLys Lys Thr Gly Lys Trp Asp 275 280 285 Ser Asn Thr Ser Thr Leu Thr IleSer Val Asn Ser Lys Lys Thr Lys 290 295 300 Asn Ile Val Phe Thr Lys GluAsp Thr Ile Thr Val Gln Lys Tyr Asp 305 310 315 320 Ser Ala Gly Thr AsnLeu Glu Gly Asn Ala Val Glu Ile Lys Thr Leu 325 330 335 Asp Glu Leu LysAsn Ala Leu Lys 340 87 1323 DNA Artificial Sequence Chimeric nucleicacid 87 atg gct tgt aat aat tca ggg aaa gat ggg aat aca tct gca aat tct48 Met Ala Cys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 510 15 gct gat gag tct gtt aaa ggg cct aat ctt aca gaa ata aat aaa aaa 96Ala Asp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30att acg gat tct aat gcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 IleThr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttgctg tca tct ata gat gaa att gct gct aaa gct att ggt aaa aaa 192 Leu LeuSer Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 ata caccaa aat aat ggt ttg gat acc gaa aat aat cac aat gga tca 240 Ile His GlnAsn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80 ttg ttagcg gga gct tat gca ata tca acc cta ata aaa caa aaa tta 288 Leu Leu AlaGly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95 gat gga ttgaaa aat gaa gga tta aag gaa aaa att gat gcg gct aag 336 Asp Gly Leu LysAsn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105 110 aaa tgt tctgaa aca ttt act aat aaa tta aaa gaa aaa cac aca gat 384 Lys Cys Ser GluThr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115 120 125 ctt ggt aaagaa ggt gtt act gat gct gat gca aaa gaa gcc att tta 432 Leu Gly Lys GluGly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu 130 135 140 aaa gca aatggt act aaa act aaa ggt gct gaa gaa ctt gga aaa tta 480 Lys Ala Asn GlyThr Lys Thr Lys Gly Ala Glu Glu Leu Gly Lys Leu 145 150 155 160 ttt gaatca gta gag gtc ttg tca aaa gca gct aaa gag atg ctt gct 528 Phe Glu SerVal Glu Val Leu Ser Lys Ala Ala Lys Glu Met Leu Ala 165 170 175 aat tcagtt aaa gag ctt aca agc cct gtt gtg gca gaa agt cca aaa 576 Asn Ser ValLys Glu Leu Thr Ser Pro Val Val Ala Glu Ser Pro Lys 180 185 190 aaa ccttcc atg gcc gtt tca gta gat ttg cct ggt gaa atg aaa gtt 624 Lys Pro SerMet Ala Val Ser Val Asp Leu Pro Gly Glu Met Lys Val 195 200 205 ctt gtaagc aaa gaa aaa aac aaa gac ggc aag tac gat cta att gca 672 Leu Val SerLys Glu Lys Asn Lys Asp Gly Lys Tyr Asp Leu Ile Ala 210 215 220 aca gtagac aag ctt gag ctt aaa gga act tct gat aaa aac aat gga 720 Thr Val AspLys Leu Glu Leu Lys Gly Thr Ser Asp Lys Asn Asn Gly 225 230 235 240 tctgga gta ctt gaa ggc gta aaa gct gac aaa agt aaa gta aaa tta 768 Ser GlyVal Leu Glu Gly Val Lys Ala Asp Lys Ser Lys Val Lys Leu 245 250 255 acaatt tct gac gat cta ggt caa acc aca ctt gaa gtt ttc aaa gaa 816 Thr IleSer Asp Asp Leu Gly Gln Thr Thr Leu Glu Val Phe Lys Glu 260 265 270 gatggc aaa aca cta gta tca aaa aaa gta act tcc aaa gac aag tca 864 Asp GlyLys Thr Leu Val Ser Lys Lys Val Thr Ser Lys Asp Lys Ser 275 280 285 tcaaca gaa gaa aaa ttc aat gaa aaa ggt gaa gta tct gaa aaa ata 912 Ser ThrGlu Glu Lys Phe Asn Glu Lys Gly Glu Val Ser Glu Lys Ile 290 295 300 ataaca aga gca gac gga acc aga ctt gaa tac aca gga att aaa agc 960 Ile ThrArg Ala Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser 305 310 315 320gat gga tct gga aaa gct aaa gag gtt tta aaa ggc ttt act ctt gaa 1008 AspGly Ser Gly Lys Ala Lys Glu Val Leu Lys Gly Phe Thr Leu Glu 325 330 335gga aaa gta gct aat gat aaa gta aca ttg gaa gta aaa gaa gga acc 1056 GlyLys Val Ala Asn Asp Lys Val Thr Leu Glu Val Lys Glu Gly Thr 340 345 350gtt act tta agt aag att tca aaa tct ggg gaa gtt tca gtt gaa ctt 1104 ValThr Leu Ser Lys Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu 355 360 365aat gac act gac agt agt gct gct act aaa aaa act gca gct tgg aat 1152 AsnAsp Thr Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn 370 375 380tca aaa act tct act tta aca att agt gtt aac agc aaa aaa act aca 1200 SerLys Thr Ser Thr Leu Thr Ile Ser Val Asn Ser Lys Lys Thr Thr 385 390 395400 caa ctt gtg ttt act aaa caa gac aca ata act gta caa aaa tac gac 1248Gln Leu Val Phe Thr Lys Gln Asp Thr Ile Thr Val Gln Lys Tyr Asp 405 410415 tcc gca ggt acc aat tta gaa ggc aca gca gtc gaa att aaa aca ctt 1296Ser Ala Gly Thr Asn Leu Glu Gly Thr Ala Val Glu Ile Lys Thr Leu 420 425430 gat gaa ctt aaa aac gct tta aaa taa 1323 Asp Glu Leu Lys Asn Ala LeuLys * 435 440 88 440 PRT Artificial Sequence Chimeric protein 88 Met AlaCys Asn Asn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 AlaAsp Glu Ser Val Lys Gly Pro Asn Leu Thr Glu Ile Asn Lys Lys 20 25 30 IleThr Asp Ser Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 LeuLeu Ser Ser Ile Asp Glu Ile Ala Ala Lys Ala Ile Gly Lys Lys 50 55 60 IleHis Gln Asn Asn Gly Leu Asp Thr Glu Asn Asn His Asn Gly Ser 65 70 75 80Leu Leu Ala Gly Ala Tyr Ala Ile Ser Thr Leu Ile Lys Gln Lys Leu 85 90 95Asp Gly Leu Lys Asn Glu Gly Leu Lys Glu Lys Ile Asp Ala Ala Lys 100 105110 Lys Cys Ser Glu Thr Phe Thr Asn Lys Leu Lys Glu Lys His Thr Asp 115120 125 Leu Gly Lys Glu Gly Val Thr Asp Ala Asp Ala Lys Glu Ala Ile Leu130 135 140 Lys Ala Asn Gly Thr Lys Thr Lys Gly Ala Glu Glu Leu Gly LysLeu 145 150 155 160 Phe Glu Ser Val Glu Val Leu Ser Lys Ala Ala Lys GluMet Leu Ala 165 170 175 Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val AlaGlu Ser Pro Lys 180 185 190 Lys Pro Ser Met Ala Val Ser Val Asp Leu ProGly Glu Met Lys Val 195 200 205 Leu Val Ser Lys Glu Lys Asn Lys Asp GlyLys Tyr Asp Leu Ile Ala 210 215 220 Thr Val Asp Lys Leu Glu Leu Lys GlyThr Ser Asp Lys Asn Asn Gly 225 230 235 240 Ser Gly Val Leu Glu Gly ValLys Ala Asp Lys Ser Lys Val Lys Leu 245 250 255 Thr Ile Ser Asp Asp LeuGly Gln Thr Thr Leu Glu Val Phe Lys Glu 260 265 270 Asp Gly Lys Thr LeuVal Ser Lys Lys Val Thr Ser Lys Asp Lys Ser 275 280 285 Ser Thr Glu GluLys Phe Asn Glu Lys Gly Glu Val Ser Glu Lys Ile 290 295 300 Ile Thr ArgAla Asp Gly Thr Arg Leu Glu Tyr Thr Gly Ile Lys Ser 305 310 315 320 AspGly Ser Gly Lys Ala Lys Glu Val Leu Lys Gly Phe Thr Leu Glu 325 330 335Gly Lys Val Ala Asn Asp Lys Val Thr Leu Glu Val Lys Glu Gly Thr 340 345350 Val Thr Leu Ser Lys Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu 355360 365 Asn Asp Thr Asp Ser Ser Ala Ala Thr Lys Lys Thr Ala Ala Trp Asn370 375 380 Ser Lys Thr Ser Thr Leu Thr Ile Ser Val Asn Ser Lys Lys ThrThr 385 390 395 400 Gln Leu Val Phe Thr Lys Gln Asp Thr Ile Thr Val GlnLys Tyr Asp 405 410 415 Ser Ala Gly Thr Asn Leu Glu Gly Thr Ala Val GluIle Lys Thr Leu 420 425 430 Asp Glu Leu Lys Asn Ala Leu Lys 435 440 891302 DNA Artificial Sequence Chimeric nucleic acid 89 atg gct tgt aataat tca ggg aaa gat ggg aat aca tct gca aat tct 48 Met Ala Cys Asn AsnSer Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 gct gat gag tctgtt aaa ggg cct aat ctt aca gaa ata agt aaa aaa 96 Ala Asp Glu Ser ValLys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 att acg gat tct aatgcg gtt tta ctt gct gtg aaa gag gtt gaa gcg 144 Ile Thr Asp Ser Asn AlaVal Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 ttg ctg tca tct ata gatgag ctt gct aaa gct att ggt aaa aaa ata 192 Leu Leu Ser Ser Ile Asp GluLeu Ala Lys Ala Ile Gly Lys Lys Ile 50 55 60 aaa aac gat ggt agt tta gataat gaa gca aat cgc aac gag tca ttg 240 Lys Asn Asp Gly Ser Leu Asp AsnGlu Ala Asn Arg Asn Glu Ser Leu 65 70 75 80 tta gca gga gct tat aca atatca acc tta ata aca caa aaa tta agt 288 Leu Ala Gly Ala Tyr Thr Ile SerThr Leu Ile Thr Gln Lys Leu Ser 85 90 95 aaa tta aac gga tca gaa ggt ttaaag gaa aag att gcc gca gct aag 336 Lys Leu Asn Gly Ser Glu Gly Leu LysGlu Lys Ile Ala Ala Ala Lys 100 105 110 aaa tgc tct gaa gag ttt agt actaaa cta aaa gat aat cat gca cag 384 Lys Cys Ser Glu Glu Phe Ser Thr LysLeu Lys Asp Asn His Ala Gln 115 120 125 ctt ggt ata cag ggc gtt act gatgaa aat gca aaa aaa gct att tta 432 Leu Gly Ile Gln Gly Val Thr Asp GluAsn Ala Lys Lys Ala Ile Leu 130 135 140 aaa gca aat gca gcg ggt aaa gataag ggc gtt gaa gaa ctt gaa aag 480 Lys Ala Asn Ala Ala Gly Lys Asp LysGly Val Glu Glu Leu Glu Lys 145 150 155 160 ttg tcc gga tca tta gaa agctta tca aaa gca gct aaa gag atg ctt 528 Leu Ser Gly Ser Leu Glu Ser LeuSer Lys Ala Ala Lys Glu Met Leu 165 170 175 gct aat tca gtt aaa gag cttaca agc cct gtt gtc cat ggc gtt tca 576 Ala Asn Ser Val Lys Glu Leu ThrSer Pro Val Val His Gly Val Ser 180 185 190 gta gat ttg cct ggt gaa atgaaa gtt ctt gta agc aaa gaa aaa aac 624 Val Asp Leu Pro Gly Glu Met LysVal Leu Val Ser Lys Glu Lys Asn 195 200 205 aaa gac ggc aag tac gat ctaatt gca aca gta gac aag ctt gag ctt 672 Lys Asp Gly Lys Tyr Asp Leu IleAla Thr Val Asp Lys Leu Glu Leu 210 215 220 aaa gga act tct gat aaa aacaat gga tct gga gta ctt gaa ggc gta 720 Lys Gly Thr Ser Asp Lys Asn AsnGly Ser Gly Val Leu Glu Gly Val 225 230 235 240 aaa gct gac aaa agt aaagta aaa tta aca att tct gac gat cta ggt 768 Lys Ala Asp Lys Ser Lys ValLys Leu Thr Ile Ser Asp Asp Leu Gly 245 250 255 caa acc aca ctt gaa gttttc aaa gaa gat ggc aaa aca cta gta tca 816 Gln Thr Thr Leu Glu Val PheLys Glu Asp Gly Lys Thr Leu Val Ser 260 265 270 aaa aaa gta act tcc aaagac aag tca tca aca gaa gaa aaa ttc aat 864 Lys Lys Val Thr Ser Lys AspLys Ser Ser Thr Glu Glu Lys Phe Asn 275 280 285 gaa aaa ggt gaa gta tctgaa aaa ata ata aca aga gca gac gga acc 912 Glu Lys Gly Glu Val Ser GluLys Ile Ile Thr Arg Ala Asp Gly Thr 290 295 300 aga ctt gaa tac aca ggaatt aaa agc gat gga tct gga aaa gct aaa 960 Arg Leu Glu Tyr Thr Gly IleLys Ser Asp Gly Ser Gly Lys Ala Lys 305 310 315 320 gag gtt tta aaa ggcttt act ctt gaa gga aaa gta gct aat gat aaa 1008 Glu Val Leu Lys Gly PheThr Leu Glu Gly Lys Val Ala Asn Asp Lys 325 330 335 gta aca ttg gaa gtaaaa gaa gga acc gtt act tta agt aag att tca 1056 Val Thr Leu Glu Val LysGlu Gly Thr Val Thr Leu Ser Lys Ile Ser 340 345 350 aaa tct ggg gaa gtttca gtt gaa ctt aat gac act gac agt agt gct 1104 Lys Ser Gly Glu Val SerVal Glu Leu Asn Asp Thr Asp Ser Ser Ala 355 360 365 gct act aaa aaa actgca gct tgg aat tca aaa act tct act tta aca 1152 Ala Thr Lys Lys Thr AlaAla Trp Asn Ser Lys Thr Ser Thr Leu Thr 370 375 380 att agt gtt aac agcaaa aaa act aca caa ctt gtg ttt act aaa caa 1200 Ile Ser Val Asn Ser LysLys Thr Thr Gln Leu Val Phe Thr Lys Gln 385 390 395 400 gac aca ata actgta caa aaa tac gac tcc gca ggt acc aat tta gaa 1248 Asp Thr Ile Thr ValGln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 405 410 415 ggc aca gca gtcgaa att aaa aca ctt gat gaa ctt aaa aac gct tta 1296 Gly Thr Ala Val GluIle Lys Thr Leu Asp Glu Leu Lys Asn Ala Leu 420 425 430 aaa taa 1302Lys * 90 433 PRT Artificial Sequence Chimeric protein 90 Met Ala Cys AsnAsn Ser Gly Lys Asp Gly Asn Thr Ser Ala Asn Ser 1 5 10 15 Ala Asp GluSer Val Lys Gly Pro Asn Leu Thr Glu Ile Ser Lys Lys 20 25 30 Ile Thr AspSer Asn Ala Val Leu Leu Ala Val Lys Glu Val Glu Ala 35 40 45 Leu Leu SerSer Ile Asp Glu Leu Ala Lys Ala Ile Gly Lys Lys Ile 50 55 60 Lys Asn AspGly Ser Leu Asp Asn Glu Ala Asn Arg Asn Glu Ser Leu 65 70 75 80 Leu AlaGly Ala Tyr Thr Ile Ser Thr Leu Ile Thr Gln Lys Leu Ser 85 90 95 Lys LeuAsn Gly Ser Glu Gly Leu Lys Glu Lys Ile Ala Ala Ala Lys 100 105 110 LysCys Ser Glu Glu Phe Ser Thr Lys Leu Lys Asp Asn His Ala Gln 115 120 125Leu Gly Ile Gln Gly Val Thr Asp Glu Asn Ala Lys Lys Ala Ile Leu 130 135140 Lys Ala Asn Ala Ala Gly Lys Asp Lys Gly Val Glu Glu Leu Glu Lys 145150 155 160 Leu Ser Gly Ser Leu Glu Ser Leu Ser Lys Ala Ala Lys Glu MetLeu 165 170 175 Ala Asn Ser Val Lys Glu Leu Thr Ser Pro Val Val His GlyVal Ser 180 185 190 Val Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser LysGlu Lys Asn 195 200 205 Lys Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val AspLys Leu Glu Leu 210 215 220 Lys Gly Thr Ser Asp Lys Asn Asn Gly Ser GlyVal Leu Glu Gly Val 225 230 235 240 Lys Ala Asp Lys Ser Lys Val Lys LeuThr Ile Ser Asp Asp Leu Gly 245 250 255 Gln Thr Thr Leu Glu Val Phe LysGlu Asp Gly Lys Thr Leu Val Ser 260 265 270 Lys Lys Val Thr Ser Lys AspLys Ser Ser Thr Glu Glu Lys Phe Asn 275 280 285 Glu Lys Gly Glu Val SerGlu Lys Ile Ile Thr Arg Ala Asp Gly Thr 290 295 300 Arg Leu Glu Tyr ThrGly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys 305 310 315 320 Glu Val LeuLys Gly Phe Thr Leu Glu Gly Lys Val Ala Asn Asp Lys 325 330 335 Val ThrLeu Glu Val Lys Glu Gly Thr Val Thr Leu Ser Lys Ile Ser 340 345 350 LysSer Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 355 360 365Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Lys Thr Ser Thr Leu Thr 370 375380 Ile Ser Val Asn Ser Lys Lys Thr Thr Gln Leu Val Phe Thr Lys Gln 385390 395 400 Asp Thr Ile Thr Val Gln Lys Tyr Asp Ser Ala Gly Thr Asn LeuGlu 405 410 415 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys AsnAla Leu 420 425 430 Lys 91 31 DNA Artificial Sequence OligonucleotidePrimer 91 gtcatatggc ttgtaataat tcagggaaag a 31 92 30 DNA ArtificialSequence Oligonucleotide Primer 92 tttccatgga aggttttttt ggactttctg 3093 30 DNA Artificial Sequence Oligonucleotide Primer 93 tttccatggccaagcaaaat gttagcagcc 30 94 27 DNA Artificial Sequence OligonucleotidePrimer 94 taaggatcct tattttaaag cgttttt 27 95 822 DNA ArtificialSequence Altered OspA Nucleic Acid 95 atg aaa aaa tat tta ttg gga ataggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile GlyLeu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc cttgac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu AspGlu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt cttgta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu ValSer Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca aca gtagac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val AspLys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct gga gtactt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val LeuGlu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca att tctgac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser AspAsp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa acacta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr LeuVal Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaaaaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu LysPhe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca atg gcagac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Met Ala AspGly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tct ggaaaa gct aaa gag 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly LysAla Lys Glu 145 150 155 160 gtt tta aaa ggc tat gtt ctt gaa gga act ctaact gct gaa aaa aca 528 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu ThrAla Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act ttaagc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu SerLys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gacact gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp ThrAsp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tca ggcact tca act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly ThrSer Thr Leu Thr 210 215 220 att act gta aac agt aaa aaa act aaa gac cttgtg ttt aca aaa gaa 720 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu ValPhe Thr Lys Glu 225 230 235 240 aac aca att aca gta caa caa tac gac tcaaat ggc acc aaa tta gag 768 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser AsnGly Thr Lys Leu Glu 245 250 255 ggg tca gca gtt gaa att aca aaa ctt gatgaa att aaa aac gct tta 816 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp GluIle Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 96 273 PRT ArtificialSequence Altered OspA protein 96 Met Lys Lys Tyr Leu Leu Gly Ile Gly LeuIle Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu AspGlu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val LeuVal Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala ThrVal Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly SerGly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys LeuThr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys GluAsp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp LysSer Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser GluLys Ile Ile Thr Met Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr GlyIle Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu LysGly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 Thr LeuVal Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 LysSer Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215220 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225230 235 240 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys LeuGlu 245 250 255 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys AsnAla Leu 260 265 270 Lys 97 822 DNA Artificial Sequence Altered OspANucleic Acid 97 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc ttaata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu IleAla 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtttca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val SerVal 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aacaaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 5055 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 6570 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 8590 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115120 125 aaa ggt gaa gta tct gaa aaa ata ata aca aga gca gac gga acc aga432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa tat480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Tyr 145150 155 160 gtt tta aaa ggc tat gtt ctt gaa gga act cta act gct gaa aaaaca 528 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaa aat atttca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agtgct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala195 200 205 gct act aaa aaa act gca gct tgg aat tca ggc act tca act ttaaca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr210 215 220 att act gta aac agt aaa aaa act aaa gac ctt gtg ttt aca aaagaa 720 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu225 230 235 240 aac aca att aca gta caa caa tac gac tca aat ggc acc aaatta gag 768 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys LeuGlu 245 250 255 ggg tca gca gtt gaa att aca aaa ctt gat gaa att aaa aacgct tta 816 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn AlaLeu 260 265 270 aaa taa 822 Lys * 98 273 PRT Artificial Sequence AlteredOspA protein 98 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala LeuIle Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn SerVal Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys GluLys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys LeuGlu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu GluGly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser AspAsp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys ThrLeu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr GluGlu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile ThrArg Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser AspGly Ser Gly Lys Ala Lys Tyr 145 150 155 160 Val Leu Lys Gly Tyr Val LeuGlu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val Val Lys GluGly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys Ser Gly Glu ValSer Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys LysThr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 Ile Thr ValAsn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 AsnThr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265270 Lys 99 822 DNA Artificial Sequence Altered OspA Nucleic Acid 99 atgaaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met LysLys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgtaag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys LysGln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttgcct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu ProGly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aagtac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys TyrAsp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gataaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp LysAsn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agtaaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser LysVal Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gttttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val PheLys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaagac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys AspLys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tctgaa aaa ata ata aca atg gca gac gga acc aga 432 Lys Gly Glu Val Ser GluLys Ile Ile Thr Met Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca ggaatt aaa agc gat gga tct gga aaa gct aaa tat 480 Leu Glu Tyr Thr Gly IleLys Ser Asp Gly Ser Gly Lys Ala Lys Tyr 145 150 155 160 gtt tta aaa ggctat gtt ctt gaa gga act cta act gct gaa aaa aca 528 Val Leu Lys Gly TyrVal Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gttaaa gaa gga act gtt act tta agc aaa aat att tca 576 Thr Leu Val Val LysGlu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tct ggg gaagtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu ValSer Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaaact gca gct tgg aat tca ggc act tca act tta aca 672 Ala Thr Lys Lys ThrAla Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 att act gta aacagt aaa aaa act aaa gac ctt gtg ttt aca aaa gaa 720 Ile Thr Val Asn SerLys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 aac aca attaca gta caa caa tac gac tca aat ggc acc aaa tta gag 768 Asn Thr Ile ThrVal Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 ggg tca gcagtt gaa att aca aaa ctt gat gaa att aaa aac gct tta 816 Gly Ser Ala ValGlu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 aaa taa 822Lys * 100 273 PRT Artificial Sequence Altered OspA Protein 100 Met LysLys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 CysLys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 AspLeu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 AspGly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 GlyThr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Met Ala Asp Gly Thr Arg130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala LysTyr 145 150 155 160 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr AlaGlu Lys Thr 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu SerLys Asn Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn AspThr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn SerGly Thr Ser Thr Leu Thr 210 215 220 Ile Thr Val Asn Ser Lys Lys Thr LysAsp Leu Val Phe Thr Lys Glu 225 230 235 240 Asn Thr Ile Thr Val Gln GlnTyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 Gly Ser Ala Val Glu IleThr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 Lys 101 822 DNAArtificial Sequence Altered OspA Nucleic Acid 101 atg aaa aaa tat ttattg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu LeuGly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gttagc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val SerSer Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atgaaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met LysVal Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta attgca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile AlaThr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat ggatct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly SerGly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa ttaaca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu ThrIle Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gatggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp GlyLys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tcaaca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser ThrGlu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ataaca aga gca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile ThrArg Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gatgga tct gga aaa gct aaa tat 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp GlySer Gly Lys Ala Lys Tyr 145 150 155 160 gtt tta aaa ggc tat gtt ctt gaagga act cta act gct gaa aaa aca 528 Val Leu Lys Gly Tyr Val Leu Glu GlyThr Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga actgtt act tta agc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr ValThr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaactt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu LeuAsn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tggaat tca ggc act tca act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp AsnSer Gly Thr Ser Thr Leu Thr 210 215 220 att act gta aac agt aaa aaa actaaa gac ctt gtg ttt aca aaa gaa 720 Ile Thr Val Asn Ser Lys Lys Thr LysAsp Leu Val Phe Thr Lys Glu 225 230 235 240 aac aca att aca gta caa caatac gac tca aat ggc acc aaa tta gag 768 Asn Thr Ile Thr Val Gln Gln TyrAsp Ser Asn Gly Thr Lys Leu Glu 245 250 255 ggg tca gca gtt gaa att acaaaa ctt gat gaa att aaa aac gct tta 816 Gly Ser Ala Val Glu Ile Thr LysLeu Asp Glu Ile Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 102 273PRT Artificial Sequence Altered OspA Protein 102 Met Lys Lys Tyr Leu LeuGly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn ValSer Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly GluMet Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr AspLeu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp LysAsn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys SerLys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu GluVal Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val ThrSer Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys GlyGlu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 LeuGlu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Tyr 145 150 155160 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser SerAla 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser ThrLeu Thr 210 215 220 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val PheThr Lys Glu 225 230 235 240 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser AsnGly Thr Lys Leu Glu 245 250 255 Gly Ser Ala Val Glu Ile Thr Lys Leu AspGlu Ile Lys Asn Ala Leu 260 265 270 Lys 103 822 DNA Artificial SequenceAltered OspA Nucleic Acid 103 atg aaa aaa tat tta ttg gga ata ggt ctaata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu IleLeu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gagaaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu LysAsn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agcaaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser LysGlu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca aca gta gac aagctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys LeuGlu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaaggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu GlyVal Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gatcta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp LeuGly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gtatca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val SerLys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttcaat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe AsnGlu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca atg gca gac ggaacc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Met Ala Asp Gly ThrArg 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gctaaa tat 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala LysTyr 145 150 155 160 gtt tta aaa ggc tat gtt ctt gaa gga act cta act gctgaa aaa aca 528 Val Leu Lys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala GluLys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaaaat att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys AsnIle Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gacagt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp SerSer Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tca ggc act tcaact tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser ThrLeu Thr 210 215 220 att act gta aac agt aaa aaa act aaa gac ctt gtg tttaca aaa gaa 720 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe ThrLys Glu 225 230 235 240 aac aca att aca gta caa caa tac gac tca aat ggcacc aaa tta gag 768 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly ThrLys Leu Glu 245 250 255 ggg tca gca gtt gaa att aca aaa ctt gat gaa attaaa aac gct tta 816 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile LysAsn Ala Leu 260 265 270 aaa taa 822 Lys * 104 273 PRT ArtificialSequence Altered OspA Protein 104 Met Lys Lys Tyr Leu Leu Gly Ile GlyLeu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser LeuAsp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys ValLeu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile AlaThr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn GlySer Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val LysLeu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe LysGlu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys AspLys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val SerGlu Lys Ile Ile Thr Met Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr ThrGly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Tyr 145 150 155 160 Val LeuLys Gly Tyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 ThrLeu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210215 220 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu225 230 235 240 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr LysLeu Glu 245 250 255 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile LysAsn Ala Leu 260 265 270 Lys 105 822 DNA Artificial Sequence Altered OspANucleic Acid 105 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc ttaata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu IleAla 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtttca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val SerVal 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aacaaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 5055 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 6570 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 8590 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115120 125 aaa ggt gaa gta tct gaa aaa ata ata aca aga gca gac gga acc aga432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa gag480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145150 155 160 gtt tta aaa ggc ttt gtt ctt gaa gga act cta act gct gaa aaaaca 528 Val Leu Lys Gly Phe Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaa aat atttca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agtgct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala195 200 205 gct act aaa aaa act gca gct tgg aat tca ggc act tca act ttaaca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr210 215 220 att act gta aac agt aaa aaa act aaa gac ctt gtg ttt aca aaagaa 720 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu225 230 235 240 aac aca att aca gta caa caa tac gac tca aat ggc acc aaatta gag 768 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys LeuGlu 245 250 255 ggg tca gca gtt gaa att aca aaa ctt gat gaa att aaa aacgct tta 816 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn AlaLeu 260 265 270 aaa taa 822 Lys * 106 273 PRT Artificial SequenceAltered OspA protein 106 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile LeuAla Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu LysAsn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val SerLys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val AspLys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly ValLeu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr IleSer Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp GlyLys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser SerThr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys IleIle Thr Arg Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile LysSer Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Gly PheVal Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val ValLys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys Ser GlyGlu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala ThrLys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 IleThr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235240 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245250 255 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu260 265 270 Lys 107 822 DNA Artificial Sequence Altered OspA NucleicAcid 107 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 510 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 AspLeu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gacggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp GlyLys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga acttct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr SerAsp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gacaaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp LysSer Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca cttgaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu GluVal Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta acttcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr SerLys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaagta tct gaa aaa ata ata aca aga gca gac gga acc aga 432 Lys Gly Glu ValSer Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 ctt gaa tacaca gga att aaa agc gat gga tct gga aaa gct aaa gag 480 Leu Glu Tyr ThrGly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt ttaaaa ggc ttt act ctt gaa gga act cta act gct gaa aaa aca 528 Val Leu LysGly Phe Thr Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 aca ttggtg gtt aaa gaa gga act gtt act tta agc aaa aat att tca 576 Thr Leu ValVal Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tctggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser GlyGlu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct actaaa aaa act gca gct tgg aat tca ggc act tca act tta aca 672 Ala Thr LysLys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 att actgta aac agt aaa aaa act aaa gac ctt gtg ttt aca aaa gaa 720 Ile Thr ValAsn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 aacaca att aca gta caa caa tac gac tca aat ggc acc aaa tta gag 768 Asn ThrIle Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 gggtca gca gtt gaa att aca aaa ctt gat gaa att aaa aac gct tta 816 Gly SerAla Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 aaataa 822 Lys * 108 273 PRT Artificial Sequence Altered OspA Protein 108Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 1015 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 2530 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 4045 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 5560 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 7075 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 8590 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe AsnGlu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp GlyThr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly LysAla Lys Glu 145 150 155 160 Val Leu Lys Gly Phe Thr Leu Glu Gly Thr LeuThr Ala Glu Lys Thr 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val ThrLeu Ser Lys Asn Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu LeuAsn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala TrpAsn Ser Gly Thr Ser Thr Leu Thr 210 215 220 Ile Thr Val Asn Ser Lys LysThr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 Asn Thr Ile Thr ValGln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 Gly Ser Ala ValGlu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 Lys 109 822DNA Artificial Sequence Altered OspA Nucleic Acid 109 atg aaa aaa tattta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr LeuLeu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aatgtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn ValSer Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaaatg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu MetLys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat ctaatt gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu IleAla Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aatgga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn GlySer Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaatta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys LeuThr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaagat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu AspGly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tcatca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser SerThr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ataata aca aga gca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile IleThr Arg Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agcgat gga tct gga aaa gct aaa gag 480 Leu Glu Tyr Thr Gly Ile Lys Ser AspGly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa ggc tat act cttgaa gga act cta act gct gaa aaa aca 528 Val Leu Lys Gly Tyr Thr Leu GluGly Thr Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa ggaact gtt act tta agc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly ThrVal Thr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gttgaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val GluLeu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gcttgg aat tca ggc act tca act tta aca 672 Ala Thr Lys Lys Thr Ala Ala TrpAsn Ser Gly Thr Ser Thr Leu Thr 210 215 220 att act gta aac agt aaa aaaact aaa gac ctt gtg ttt aca aaa gaa 720 Ile Thr Val Asn Ser Lys Lys ThrLys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 aac aca att aca gta caacaa tac gac tca aat ggc acc aaa tta gag 768 Asn Thr Ile Thr Val Gln GlnTyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 ggg tca gca gtt gaa attaca aaa ctt gat gaa att aaa aac gct tta 816 Gly Ser Ala Val Glu Ile ThrLys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 110273 PRT Artificial Sequence Altered OspA Protein 110 Met Lys Lys Tyr LeuLeu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln AsnVal Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro GlyGlu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys TyrAsp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser AspLys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp LysSer Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr LeuGlu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys ValThr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 LysGly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150155 160 Val Leu Lys Gly Tyr Thr Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn IleSer 180 185 190 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp SerSer Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr SerThr Leu Thr 210 215 220 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu ValPhe Thr Lys Glu 225 230 235 240 Asn Thr Ile Thr Val Gln Gln Tyr Asp SerAsn Gly Thr Lys Leu Glu 245 250 255 Gly Ser Ala Val Glu Ile Thr Lys LeuAsp Glu Ile Lys Asn Ala Leu 260 265 270 Lys 111 822 DNA ArtificialSequence Altered OspA Nucleic Acid 111 atg aaa aaa tat tta ttg gga ataggt cta ata tta gcc tta ata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile GlyLeu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caa aat gtt agc agc cttgac gag aaa aac agc gtt tca gta 96 Cys Lys Gln Asn Val Ser Ser Leu AspGlu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggt gaa atg aaa gtt cttgta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu ValSer Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gat cta att gca aca gtagac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val AspLys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aac aat gga tct gga gtactt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val LeuGlu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gta aaa tta aca att tctgac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser AspAsp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa acacta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr LeuVal Ser Lys 100 105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaaaaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu LysPhe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaa ata ata aca aga gcagac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala AspGly Thr Arg 130 135 140 ctt gaa tac aca gga att aaa agc gat gga tct ggaaaa gct aaa gag 480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly LysAla Lys Glu 145 150 155 160 gtt tta aaa ggc tat act ctt gaa gga aag ctaact gct gaa aaa aca 528 Val Leu Lys Gly Tyr Thr Leu Glu Gly Lys Leu ThrAla Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act ttaagc aaa aat att tca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu SerLys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gacact gac agt agt gct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp ThrAsp Ser Ser Ala 195 200 205 gct act aaa aaa act gca gct tgg aat tca ggcact tca act tta aca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly ThrSer Thr Leu Thr 210 215 220 att act gta aac agt aaa aaa act aaa gac cttgtg ttt aca aaa gaa 720 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu ValPhe Thr Lys Glu 225 230 235 240 aac aca att aca gta caa caa tac gac tcaaat ggc acc aaa tta gag 768 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser AsnGly Thr Lys Leu Glu 245 250 255 ggg tca gca gtt gaa att aca aaa ctt gatgaa att aaa aac gct tta 816 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp GluIle Lys Asn Ala Leu 260 265 270 aaa taa 822 Lys * 112 273 PRT ArtificialSequence Altered OspA Protein 112 Met Lys Lys Tyr Leu Leu Gly Ile GlyLeu Ile Leu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser LeuAsp Glu Lys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys ValLeu Val Ser Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile AlaThr Val Asp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn GlySer Gly Val Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val LysLeu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe LysGlu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys AspLys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val SerGlu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr ThrGly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val LeuLys Gly Tyr Thr Leu Glu Gly Lys Leu Thr Ala Glu Lys Thr 165 170 175 ThrLeu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200205 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210215 220 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu225 230 235 240 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr LysLeu Glu 245 250 255 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile LysAsn Ala Leu 260 265 270 Lys 113 822 DNA Artificial Sequence Altered OspANucleic Acid 113 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc ttaata gca 48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu IleAla 1 5 10 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtttca gta 96 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val SerVal 20 25 30 gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aacaaa 144 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys35 40 45 gac ggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa192 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 5055 60 gga act tct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa240 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 6570 75 80 gct gac aaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa288 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 8590 95 acc aca ctt gaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa336 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100105 110 aaa gta act tcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa384 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115120 125 aaa ggt gaa gta tct gaa aaa ata ata aca aga gca gac gga acc aga432 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Arg Ala Asp Gly Thr Arg 130135 140 ctt gaa tac aca gga att aaa agc gat gga tct gga aaa gct aaa gag480 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145150 155 160 gtt tta aaa ggc ttt act ctt gaa gga aag cta act gct gaa aaaaca 528 Val Leu Lys Gly Phe Thr Leu Glu Gly Lys Leu Thr Ala Glu Lys Thr165 170 175 aca ttg gtg gtt aaa gaa gga act gtt act tta agc aaa aat atttca 576 Thr Leu Val Val Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser180 185 190 aaa tct ggg gaa gtt tca gtt gaa ctt aat gac act gac agt agtgct 624 Lys Ser Gly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala195 200 205 gct act aaa aaa act gca gct tgg aat tca ggc act tca act ttaaca 672 Ala Thr Lys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr210 215 220 att act gta aac agt aaa aaa act aaa gac ctt gtg ttt aca aaagaa 720 Ile Thr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu225 230 235 240 aac aca att aca gta caa caa tac gac tca aat ggc acc aaatta gag 768 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys LeuGlu 245 250 255 ggg tca gca gtt gaa att aca aaa ctt gat gaa att aaa aacgct tta 816 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn AlaLeu 260 265 270 aaa taa 822 Lys * 114 273 PRT Artificial SequenceAltered OspA protein 114 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile LeuAla Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu LysAsn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu Val SerLys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val AspLys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly ValLeu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu Thr IleSer Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu Asp GlyLys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys Ser SerThr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu Lys IleIle Thr Arg Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile LysSer Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys Gly PheThr Leu Glu Gly Lys Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu Val ValLys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys Ser GlyGlu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 Ala ThrLys Lys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 IleThr Val Asn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235240 Asn Thr Ile Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245250 255 Gly Ser Ala Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu260 265 270 Lys 115 822 DNA Artificial Sequence Altered OspA NucleicAcid 115 atg aaa aaa tat tta ttg gga ata ggt cta ata tta gcc tta ata gca48 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 510 15 tgt aag caa aat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30gat ttg cct ggt gaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 AspLeu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gacggc aag tac gat cta att gca aca gta gac aag ctt gag ctt aaa 192 Asp GlyLys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga acttct gat aaa aac aat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr SerAsp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gacaaa agt aaa gta aaa tta aca att tct gac gat cta ggt caa 288 Ala Asp LysSer Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca cttgaa gtt ttc aaa gaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu GluVal Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta acttcc aaa gac aag tca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr SerLys Asp Lys Ser Ser Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaagta tct gaa aaa ata ata aca atg gca gac gga acc aga 432 Lys Gly Glu ValSer Glu Lys Ile Ile Thr Met Ala Asp Gly Thr Arg 130 135 140 ctt gaa tacaca gga att aaa agc gat gga tct gga aaa gct aaa tat 480 Leu Glu Tyr ThrGly Ile Lys Ser Asp Gly Ser Gly Lys Ala Lys Tyr 145 150 155 160 gtt ttaaaa ggc ttt act ctt gaa gga aag cta act gct gaa aaa aca 528 Val Leu LysGly Phe Thr Leu Glu Gly Lys Leu Thr Ala Glu Lys Thr 165 170 175 aca ttggtg gtt aaa gaa gga act gtt act tta agc atg aat att tca 576 Thr Leu ValVal Lys Glu Gly Thr Val Thr Leu Ser Met Asn Ile Ser 180 185 190 aaa tctggg gaa gtt tca gtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser GlyGlu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct actaaa aaa act gca gct tgg aat tca ggc act tca act tta aca 672 Ala Thr LysLys Thr Ala Ala Trp Asn Ser Gly Thr Ser Thr Leu Thr 210 215 220 att actgta aac agt aaa aaa act aaa gac ctt gtg ttt aca aaa gaa 720 Ile Thr ValAsn Ser Lys Lys Thr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 aacaca att aca gta caa caa tac gac tca aat ggc acc aaa tta gag 768 Asn ThrIle Thr Val Gln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 gggtca gca gtt gaa att aca aaa ctt gat gaa att aaa aac gct tta 816 Gly SerAla Val Glu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 aaataa 822 Lys * 116 273 PRT Artificial Sequence Altered OspA protein 116Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 1015 Cys Lys Gln Asn Val Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 2530 Asp Leu Pro Gly Glu Met Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 4045 Asp Gly Lys Tyr Asp Leu Ile Ala Thr Val Asp Lys Leu Glu Leu Lys 50 5560 Gly Thr Ser Asp Lys Asn Asn Gly Ser Gly Val Leu Glu Gly Val Lys 65 7075 80 Ala Asp Lys Ser Lys Val Lys Leu Thr Ile Ser Asp Asp Leu Gly Gln 8590 95 Thr Thr Leu Glu Val Phe Lys Glu Asp Gly Lys Thr Leu Val Ser Lys100 105 110 Lys Val Thr Ser Lys Asp Lys Ser Ser Thr Glu Glu Lys Phe AsnGlu 115 120 125 Lys Gly Glu Val Ser Glu Lys Ile Ile Thr Met Ala Asp GlyThr Arg 130 135 140 Leu Glu Tyr Thr Gly Ile Lys Ser Asp Gly Ser Gly LysAla Lys Tyr 145 150 155 160 Val Leu Lys Gly Phe Thr Leu Glu Gly Lys LeuThr Ala Glu Lys Thr 165 170 175 Thr Leu Val Val Lys Glu Gly Thr Val ThrLeu Ser Met Asn Ile Ser 180 185 190 Lys Ser Gly Glu Val Ser Val Glu LeuAsn Asp Thr Asp Ser Ser Ala 195 200 205 Ala Thr Lys Lys Thr Ala Ala TrpAsn Ser Gly Thr Ser Thr Leu Thr 210 215 220 Ile Thr Val Asn Ser Lys LysThr Lys Asp Leu Val Phe Thr Lys Glu 225 230 235 240 Asn Thr Ile Thr ValGln Gln Tyr Asp Ser Asn Gly Thr Lys Leu Glu 245 250 255 Gly Ser Ala ValGlu Ile Thr Lys Leu Asp Glu Ile Lys Asn Ala Leu 260 265 270 Lys 117 30DNA Artificial Sequence Oligonucleotide Primer 117 gaaaaaataa taacaatggcagacggaacc 30 118 30 DNA Artificial Sequence Oligonucleotide Primer 118ggttccgtct gccattgtta ttattttttc 30 119 27 DNA Artificial SequenceOligonucleotide Primer 119 ggaaaagcta aatatgtttt aaaaggc 27 120 27 DNAArtificial Sequence Oligonucleotide Primer 120 gccttttaaa acatatttagcttttcc 27 121 29 DNA Artificial Sequence Oligonucleotide Primer 121gttactttaa gcatgaatat ttcaaaatc 29 122 29 DNA Artificial SequenceOligonucleotide Primer 122 gattttgaaa tattcatgct taaagtaac 29 123 34 DNAArtificial Sequence Oligonucleotide Primer 123 gaggttttaa aaggctttactcttgaagga actc 34 124 34 DNA Artificial Sequence Oligonucleotide Primer124 gagttccttc aagagtaaag ccttttaaaa cctg 34 125 23 DNA ArtificialSequence Oligonucleotide Primer 125 tcttgaagga aagctaactg ctg 23 126 23DNA Artificial Sequence Oligonucleotide Primer 126 cagcagttag ctttccttcaaga 23 127 819 DNA Borrelia burgdorferi CDS (1)...(819) 127 atg aaa aaatat tta ttg gga ata ggt cta ata tta gcc tta ata gca 48 Met Lys Lys TyrLeu Leu Gly Ile Gly Leu Ile Leu Ala Leu Ile Ala 1 5 10 15 tgt aag caaaat gtt agc agc ctt gac gag aaa aac agc gtt tca gta 96 Cys Lys Gln AsnVal Ser Ser Leu Asp Glu Lys Asn Ser Val Ser Val 20 25 30 gat ttg cct ggtgaa atg aaa gtt ctt gta agc aaa gaa aaa aac aaa 144 Asp Leu Pro Gly GluMet Lys Val Leu Val Ser Lys Glu Lys Asn Lys 35 40 45 gac ggc aag tac gatcta att gca aca gta gac aag ctt gag ctt aaa 192 Asp Gly Lys Tyr Asp LeuIle Ala Thr Val Asp Lys Leu Glu Leu Lys 50 55 60 gga act tct gat aaa aacaat gga tct gga gta ctt gaa ggc gta aaa 240 Gly Thr Ser Asp Lys Asn AsnGly Ser Gly Val Leu Glu Gly Val Lys 65 70 75 80 gct gac aaa agt aaa gtaaaa tta aca att tct gac gat cta ggt caa 288 Ala Asp Lys Ser Lys Val LysLeu Thr Ile Ser Asp Asp Leu Gly Gln 85 90 95 acc aca ctt gaa gtt ttc aaagaa gat ggc aaa aca cta gta tca aaa 336 Thr Thr Leu Glu Val Phe Lys GluAsp Gly Lys Thr Leu Val Ser Lys 100 105 110 aaa gta act tcc aaa gac aagtca tca aca gaa gaa aaa ttc aat gaa 384 Lys Val Thr Ser Lys Asp Lys SerSer Thr Glu Glu Lys Phe Asn Glu 115 120 125 aaa ggt gaa gta tct gaa aaaata ata aca aga gca gac gga acc aga 432 Lys Gly Glu Val Ser Glu Lys IleIle Thr Arg Ala Asp Gly Thr Arg 130 135 140 ctt gaa tac aca gga att aaaagc gat gga tct gga aaa gct aaa gag 480 Leu Glu Tyr Thr Gly Ile Lys SerAsp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 gtt tta aaa ggc tat gttctt gaa gga act cta act gct gaa aaa aca 528 Val Leu Lys Gly Tyr Val LeuGlu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 aca ttg gtg gtt aaa gaagga act gtt act tta agc aaa aat att tca 576 Thr Leu Val Val Lys Glu GlyThr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 aaa tct ggg gaa gtt tcagtt gaa ctt aat gac act gac agt agt gct 624 Lys Ser Gly Glu Val Ser ValGlu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 gct act aaa aaa act gcagct tgg aat gca ggc act tca act tta aca 672 Ala Thr Lys Lys Thr Ala AlaTrp Asn Ala Gly Thr Ser Thr Leu Thr 210 215 220 att act gta aac aac aaaaaa act aaa gcc ctt gta ttt aca aaa caa 720 Ile Thr Val Asn Asn Lys LysThr Lys Ala Leu Val Phe Thr Lys Gln 225 230 235 240 gac aca att aca tcacaa aaa tac gac tca gca gga acc aac ttg gaa 768 Asp Thr Ile Thr Ser GlnLys Tyr Asp Ser Ala Gly Thr Asn Leu Glu 245 250 255 ggc aca gca gtc gaaatt aaa aca ctt gat gaa ctt aaa aac gct tta 816 Gly Thr Ala Val Glu IleLys Thr Leu Asp Glu Leu Lys Asn Ala Leu 260 265 270 aga 819 Arg 128 273PRT Borrelia burgdorferi 128 Met Lys Lys Tyr Leu Leu Gly Ile Gly Leu IleLeu Ala Leu Ile Ala 1 5 10 15 Cys Lys Gln Asn Val Ser Ser Leu Asp GluLys Asn Ser Val Ser Val 20 25 30 Asp Leu Pro Gly Glu Met Lys Val Leu ValSer Lys Glu Lys Asn Lys 35 40 45 Asp Gly Lys Tyr Asp Leu Ile Ala Thr ValAsp Lys Leu Glu Leu Lys 50 55 60 Gly Thr Ser Asp Lys Asn Asn Gly Ser GlyVal Leu Glu Gly Val Lys 65 70 75 80 Ala Asp Lys Ser Lys Val Lys Leu ThrIle Ser Asp Asp Leu Gly Gln 85 90 95 Thr Thr Leu Glu Val Phe Lys Glu AspGly Lys Thr Leu Val Ser Lys 100 105 110 Lys Val Thr Ser Lys Asp Lys SerSer Thr Glu Glu Lys Phe Asn Glu 115 120 125 Lys Gly Glu Val Ser Glu LysIle Ile Thr Arg Ala Asp Gly Thr Arg 130 135 140 Leu Glu Tyr Thr Gly IleLys Ser Asp Gly Ser Gly Lys Ala Lys Glu 145 150 155 160 Val Leu Lys GlyTyr Val Leu Glu Gly Thr Leu Thr Ala Glu Lys Thr 165 170 175 Thr Leu ValVal Lys Glu Gly Thr Val Thr Leu Ser Lys Asn Ile Ser 180 185 190 Lys SerGly Glu Val Ser Val Glu Leu Asn Asp Thr Asp Ser Ser Ala 195 200 205 AlaThr Lys Lys Thr Ala Ala Trp Asn Ala Gly Thr Ser Thr Leu Thr 210 215 220Ile Thr Val Asn Asn Lys Lys Thr Lys Ala Leu Val Phe Thr Lys Gln 225 230235 240 Asp Thr Ile Thr Ser Gln Lys Tyr Asp Ser Ala Gly Thr Asn Leu Glu245 250 255 Gly Thr Ala Val Glu Ile Lys Thr Leu Asp Glu Leu Lys Asn AlaLeu 260 265 270 Arg 129 9 PRT Artificial Sequence Residue 165-173 of B31OspA 129 Tyr Val Leu Glu Gly Thr Leu Thr Ala 1 5 130 9 PRT ArtificialSequence Residue 332-340 of hLFA-1 130 Tyr Val Ile Glu Gly Thr Ser LysGln 1 5

What is claimed is:
 1. A polypeptide, comprising an amino acid sequenceof OspA protein from Borrelia burgdorferi from about residue 139 toabout residue 273 of Borrelia burgdorferi OspA protein, wherein thesequence includes at least one alteration selected from the groupconsisting of: residue 139 being methionine, residue 160 being tyrosine,residue 189 being methionine and combinations thereof, wherein thenumbering corresponds to the numbering of SEQ ID NO:
 7. 2. Thepolypeptide of claim 1, wherein the polypeptide has increasedconformational stability compared to the corresponding unaltered OspApolypeptide.
 3. The polypeptide of claim 1, wherein residue 160 istyrosine and residue 189 is methionine.
 4. The polypeptide of claim 1,wherein residue 139 is methionine, residue 160 is tyrosine and residue189 is methionine.
 5. The polypeptide of claim 1, wherein thepolypeptide comprises residues 131 through 273 of the Borreliaburgdorferi OspA protein.
 6. The polypeptide of claim 1, wherein thepolypeptide comprises residues 17 through 273 of the Borreliaburgdorferi OspA protein.
 7. The polypeptide of claim 1, wherein thepolypeptide is derived from an OspA protein of a sensu stricto strain ofBorrelia burgdorferi.
 8. A polypeptide, comprising an amino acidsequence of OspA protein from a sensu stricto strain of Borreliaburgdorferi from about residue 160 to about residue 170, wherein thesequence includes at least one alteration selected from the groupconsisting of: residue 165 being phenylalanine, residue 166 beingthreonine, residue 170 being lysine and combinations thereof, whereinthe numbering corresponds to the numbering of SEQ ID NO:
 7. 9. Thepolypeptide of claim 8, wherein cross-reactivity to an hLFA-1 moleculeis reduced compared to the corresponding unaltered OspA polypeptide. 10.The polypeptide of claim 8, wherein residue 165 is phenylalanine andresidue 166 is threonine.
 11. The polypeptide of claim 8, whereinresidue 165 is phenylalanine, residue 166 is threonine and residue 170is lysine.
 12. The polypeptide of claim 8, wherein residue 139 ismethionine, residue 160 is tyrosine and residue 189 is methionine. 13.The polypeptide of claim 8, wherein the polypeptide comprises residues150 through 180 of the Borrelia burgdorferi OspA protein.
 14. Thepolypeptide of claim 8, wherein the polypeptide comprises residues 17through 273 of the Borrelia burgdorferi OspA protein.
 15. A polypeptideselected from the group consisting of: SEQ ID NO: 96, 98, 100, 102, 104,106, 108, 110, 112, 114 and
 116. 16. A polynucleotide encoding an aminoacid sequence of an OspA protein from Borrelia burgdorferi from aboutresidue 139 to about residue 273, wherein the sequence encodes at leastone alteration selected from the group consisting of: codon 139 encodingmethionine, codon 160 encoding tyrosine, codon 189 encoding methionineand combinations thereof, wherein the numbering corresponds to thenumbering of SEQ ID NO:
 7. 17. The polynucleotide of claim 16, whereincodon 160 encodes tyrosine and codon 189 encodes methionine.
 18. Thepolynucleotide of claim 16, wherein codon 139 encodes methionine, codon160 encodes tyrosine and codon 189 encodes methionine.
 19. Thepolynucleotide of claim 16, wherein the encoded polypeptide comprisesresidues 131 through 273 of the Borrelia burgdorferi OspA protein. 20.The polynucleotide of claim 16, wherein the encoded polypeptidecomprises residues 17 through 273 of the Borrelia burgdorferi OspAprotein.
 21. The polynucleotide of claim 16, wherein the encodedpolypeptide corresponds to OspA protein of a sensu stricto strain ofBorrelia burgdorferi.
 22. A polynucleotide encoding an amino acidsequence of an OspA protein from Borrelia burgdorferi from about residue160 to about residue 170, wherein the sequence encodes at least onealteration selected from the group consisting of: codon 165 encodingphenylalanine, codon 166 encoding threonine, codon 170 encoding lysineand combinations thereof, wherein the numbering corresponds to thenumbering of SEQ ID NO:
 7. 23. The polynucleotide of claim 22, whereincodon 165 encodes phenylalanine and codon 166 encodes threonine.
 24. Thepolynucleotide of claim 22, wherein codon 165 encodes phenylalanine,codon 166 encodes threonine and codon 170 encodes lysine.
 25. Apolynucleotide selected from the group consisting of: SEQ ID NO: 95, 97,99, 101, 103, 105, 107, 109, 111, 113 and
 115. 26. A method ofgenerating an altered Borrelia burgdorferi OspA polypeptide withincreased conformational stability compared to the correspondingunaltered Borrelia burgdorferi OspA polypeptide comprising; a) selectinga polynucleotide encoding a Borrelia burgdorferi OspA polypeptide thatincludes residues 139, 160 and 189, wherein the numbering corresponds tothe numbering of SEQ ID NO: 7; b) altering the polynucleotide such thatresidue 139 is methionine, residue 160 is tyrosine and residue 189 ismethionine; and c) expressing said altered polynucleotide; therebygenerating an altered Borrelia burgdorferi OspA polypeptide withincreased conformational stability compared to the correspondingunaltered Borrelia burgdorferi OspA polypeptide.
 27. A method ofgenerating an altered Borrelia burgdorferi OspA polypeptide with reducedcross-reactivity with an hLFA-1 molecule, compared to the correspondingunaltered Borrelia burgdorferi OspA polypeptide, comprising; a)selecting a polynucleotide encoding an OspA polypeptide from Borreliaburgdorferi that includes residues 165, 166 and 170, wherein thenumbering corresponds to the numbering of SEQ ID NO: 7; b) altering thepolynucleotide such that residue 165 is phenylalanine, residue 166 isthreonine and residue 170 is lysine; and c) expressing said alteredpolynucleotide; thereby generating an altered Borrelia burgdorferi OspApolypeptide with reduced cross-reactivity with the hLFA-1 moleculecompared to the corresponding unaltered Borrelia burgdorferi OspApolypeptide.
 28. Use of an altered OspA polypeptide for the manufactureof a medicament for immunizing a mammal, wherein the altered OspApolypeptide comprises: a) an amino acid sequence of OspA protein fromBorrelia burgdorferi from about residue 139 to about residue 273 ofBorrelia burgdorferi OspA protein, wherein the sequence includes atleast one alteration selected from the group consisting of: residue 139being methionine, residue 160 being tyrosine, residue 189 beingmethionine and combinations thereof, wherein the polypeptide hasincreased conformational stability compared to a corresponding wild typeOspA polypeptide; and/or b) an amino acid sequence of OspA protein fromBorrelia burgdorferi from about residue 160 to about residue 170,wherein the sequence includes at least one alteration selected from thegroup consisting of: residue 165 being phenylalanine, residue 166 beingthreonine, residue 170 being lysine and combinations thereof, whereinthe polypeptide has decreased cross-reactivity to hLFA-1 compared to acorresponding wild type OspA polypeptide; wherein the numberingcorresponds to the numbering of SEQ ID NO:
 7. 29. A polypeptide,comprising an amino acid sequence of OspA protein from Borreliaburgdorferi from about residue 160 to about residue 170, wherein thesequence includes at least two alterations selected from the groupconsisting of: residue 165 being phenylalanine, residue 166 beingthreonine and residue 170 being lysine, wherein the numberingcorresponds to the numbering of SEQ ID NO:
 7. 30. A chimericpolypeptide, comprising: a) an amino acid sequence of a first OspApolypeptide from about residue 1 to about residue 164 from a firststrain of Borrelia burgdorferi; b) an amino acid sequence of a secondOspA polypeptide from about residue 165 to about residue 179 from asecond strain of Borrelia burgdorferi, wherein said second strain is adifferent strain from said first strain; c) an amino acid sequence of athird OspA polypeptide from about residue 180 to about residue 216 froma third strain of Borrelia burgdorferi, wherein said third strain is adifferent strain from said second strain; d) an amino acid sequence of afourth OspA polypeptide from about residue 217 to about residue 273 froma fourth strain of Borrelia burgdorferi, wherein said fourth strain is adifferent strain from said third strain; wherein the sequence includesat least one alteration selected from the group consisting of: residue139 being methionine, residue 160 being tyrosine, residue 189 beingmethionine and combinations thereof, wherein the numbering correspondsto the numbering of SEQ ID NO: 7.